Synthesis of fatty acids

ABSTRACT

The present invention relates to enzymes which possess desaturase, conjugase, epoxidase and/or hydroxylase activity that can be used in methods of synthesizing fatty acids.

FIELD OF THE INVENTION

The present invention relates to enzymes which possess desaturase,conjugase, epoxidase, and/or hydroxylase activity that can be used inmethods of synthesizing fatty acids.

BACKGROUND OF THE INVENTION

The primary products of fatty acid biosynthesis in most organisms are16- and 18-carbon compounds. However, the relative ratio of chainlengths and degree of unsaturation of these fatty acids vary widelyamong species. Mammals and insects, for example, produce primarilysaturated and monosaturated fatty acids, while most higher plantsproduce fatty acids with one, two, or three double bonds, the latter twocomprising polyunsaturated fatty acids (PUFA's).

Two main families of PUFAs are the omega-3 fatty acids (also representedas “n-3” fatty acids), exemplified by eicosapentaenoic acid (EPA, 20:5,n-3), and the omega-6 fatty acids (also represented as “n-6” fattyacids), exemplified by arachidonic acid (ARA, 20:4, n-6). PUFAs areimportant components of the plasma membranes of cells, predominantlyesterified in the form of phospholipids, and adipose tissue intriglycerides.

The ability of cells to modulate the degree of unsaturation in theirmembranes is mainly determined by the action of fatty acid desaturases.Desaturase enzymes introduce unsaturated bonds at specific positions intheir fatty acyl chain substrates. Desaturase enzymes generally showconsiderable selectivity both for the chain length of the substrate andfor the location of existing double bonds in the fatty acyl chain(Shanklin and Cahoon, 1998) and may be classified on this basis. Anotherclassification of fatty acid desaturases is based on the moiety to whichthe hydrocarbon chains of their substrates are acylated. Desaturasesrecognize substrates that are bound either to acyl carrier protein, tocoenzyme A, or to lipid molecules such as phospholipids (Murata andWada, 1995; Shanklin and Cahoon, 1998).

The desaturation of fatty acids in glycerolipids is essential for theproper function of biological membranes. Introduction of unsaturation inthe Δ9 position of palmitic or stearic acid provides fluidity tomembrane lipids and thus Δ9 desaturases are found universally in livingsystems.

Linoleic acid (LA; 18:2, Δ9, 12) is produced from oleic acid (18:1, Δ9)by a Δ12-desaturase while α-linolenic acid (ALA; 18:3) is produced fromLA acid by a Δ15-desaturase. Stearidonic acid (18:4, Δ6, 9, 12, 15) andγ-linolenic acid (18:3, Δ6, 9, 12) are produced from ALA and LA,respectively, by a Δ6-desaturase. However, mammals cannot desaturatebeyond the Δ9 position and therefore cannot convert oleic acid into LA.Fourteen insect species (de Renobales, 1987) have been shown to producelinoleic acid de novo from ¹⁴C-acetate, suggesting the presence ofnative Δ12-desaturase activity. The house cricket Acheta domesticusΔ12-desaturase activity was the first of this type reported to utiliseoleoyl-CoA (Cripps, 1990). However, no gene responsible for theconversion of oleic acid to LA has been identified from an insectdespite extensive effort. Likewise, ALA cannot be synthesized bymammals. The major poly-unsaturated fatty acids of animals therefore arederived from diet via the subsequent desaturation and elongation ofdietary LA and ALA. Other eukaryotes, including fungi, nematodes andplants, have enzymes which desaturate at the carbon 12 and carbon 15positions. The membrane-associated Δ12 desaturases of Arabidopsis sp.and soybean use acyl lipid substrates

Less commonly, saturated fatty acids can be unsaturated initially atpositions other than the 9-position by desaturases with unusualspecificities. Petroselinic acid (cis-6-octadecenoic acid) isconcentrated in the seed oils of the Umbelliferae (Apiaceae), Araliaceaeand Garryaceae plant families, where it can reach 85% of the total lipidfatty acid (Kleiman and Spencer, 1982). The desaturase from coriander(Coriandrum sativum) has been characterised that makes the unusuallipid. A plastid-located acyl-acyl carrier protein (ACP) Δ4 desaturaseacts on ACP-palmitic acid to produce cis-4-hexadecenoic acid which istransferred from the plastid to the developing seed where it iselongated to cis-6-octadecenoic acid (Cahoon et al. 1992). A relateddesaturase with 83% sequence identity has been obtained from English Ivy(Hedra helix L.) which produces cis-4-hexadecenoic acid andcis-6-octadecenoic acid when expressed in Arabidopsis (Whittle et al2005). cis-5-Eicosenoic acid (C20:1 Δ5) is a major component of the seedoil of meadowfoam (Limnanthes alba) and related Limnathes species. Theenzyme responsible for the production of the unusual oil in L. douglasiiwas identified as an acyl coenzyme A-Δ5 desaturase whose substratepreference is eicosanoic acid (Cahoon et al., 2000). Expression of L.douglasii Δ5 desaturase and fatty acid elongase genes in soybean embryosresulted in the production of cis-5-eicosenoic acid (C20:1 Δ5) andcis-5-docosenoic acid (C22:1 Δ5). Sayanova et al (2007) have identifiedan acyl CoA-Δ5 desaturase which is related to the Limnathes sp.desaturase but it utilizes saturated fatty acids (C16:0, C18:0) andunsaturated fatty acids (LA, ALA) to make Δ5 monoenoic acids andpolyunsaturated fatty acids. Genes encoding similar enzymes have notbeen cloned from animals such as insects.

Some Lepidopteran insects carry out Δ11 desaturation of saturated fattyacids (C16:0, C18:0) esterified to acylCoA as a step in the productionof a diversity of moth sex pheromones (Rodriguez et al 2004).Desaturases from the moth Spodoptera littoralis were expressed inSaccharomyces cerevisiae to produce cis-Δ11 mono-unsaturated products ofC14:0, C16:0 and C18:0 when the yeast cells were fed additionalsaturated fatty acids (Rodriguez et al 2004). In addition, trans-Δ11tetradecenoic acid was formed from myristic acid (C14:0) fed to theyeast. A minor byproduct of the Δ11 desaturation was the formation of11-hydroxy hexadecanoic or octadecanoic acid (up to 0.1% of total fattyacids) (Serra et al., 2006). Moto et al. (2004) identified abi-functional acyl-CoA desaturase from the pheromone gland of thesilkmoth which was responsible for the biosynthesis of the pheromoneprecursor. The desaturase first utilised palmitic acid to makecis-11-hexadecenoic acid and then acted on this to remove allylic 2H andform a conjugated diene fatty acid (trans-Δ10,cis-Δ12-hexadecendienoicacid and some trans-Δ10,trans-Δ12-hexadecendienoic acid) and thereforeit possessed both cis-Δ11 and conjugase desaturase activities. In theNew Zealand leaf roller, Planotortrix octo, a desaturase has beenidentified from pheromone gland that desaturates palmitic acid at the MOposition to form cis-MO-hexadecenoic acid (Hao et al. 2002).

Omega-3 LC-PUFA are now widely recognized as important compounds forhuman and animal health and the inclusion of omega-3 LC-PUFA such as EPAand DHA in the human diet has been linked with numerous health-relatedbenefits. These include prevention or reduction of coronary heartdisease, hypertension, type-2 diabetes, renal disease, rheumatoidarthritis, ulcerative colitis, chronic obstructive pulmonary disease,various mental disorders such as schizophrenia, attention deficithyperactive disorder and Alzheimer's disease, and aiding braindevelopment and growth (Simopoulos, 2000). These fatty acids may beobtained from dietary sources or by conversion of linoleic (LA, omega-6)or α-linolenic (ALA, omega-3) fatty acids, both of which are regarded asessential fatty acids in the human diet. While humans and many othervertebrate animals are able to convert LA or ALA, obtained from plantsources, to LC-PUFA, they carry out this conversion at a very low rate.Moreover, most modern societies have imbalanced diets in which at least90% of polyunsaturated fatty acid(s) consist of omega-6 fatty acids,instead of the 4:1 ratio or less for omega-6:omega-3 fatty acids that isregarded as ideal (Trautwein, 2001). The immediate dietary source ofLC-PUFA such as eicosapentaenoic acid (EPA, 20:5) and docosahexaenoicacid (DHA, 22:6) for humans is mostly from fish or fish oil. Healthprofessionals have therefore recommended the regular inclusion of fishcontaining significant levels of LC-PUFA into the human diet.Increasingly, fish-derived LC-PUFA oils are being incorporated into foodproducts and in infant formula. However, due to a decline in global andnational fisheries, alternative sources of these beneficialhealth-enhancing oils are needed.

Fatty acids may also be hydroxylated, for example ricinoleic acid(12-hydroxy-octadec-cis-9-enoic acid) which comprises up to 90% of thefatty acid in castor oil from Ricinus communis and is an importantagricultural commodity oil. Other related hydroylated fatty acids foundin plant oils include 12-hydroxy-octadeca-cis-9,cis-15-dienoic(densipolic) and 14-hydroxy-eicosa-cis-11,cis-17-dienoic (auricolic)acids. The Ricinus Δ12 hydroxylase acts on oleic acid lipid substrate toproduce ricinoleic acid; the desaturase gene responsible for thetransformation is most closely related to but divergent from plantmembrane Δ12 acyl lipid desaturases (van de Loo et al 1995). There is ahomologous C20 hydroxylated fatty acid produced at high levels in seedoil of Lesquerella sp. as 14-hydroxy-eicos-cis-11-enoic (lesquerolicacid) (Gunstone et al., 1994). The L. fendleri hydroxylase gene has beencloned and expressed in an Arabidopsis FAD2 mutant which accumulatedricinoleic, lesquerolic and densipolic acids in seeds (Brow et al 1998).2-hydroxy fatty acids occur in appreciable amounts in the sphingolipidsof plants and animals but they are also present as minor components ofseeds oils such as 2-hydroxy-octadeca-9,12,15-trienoate from Thymusvulgaris seeds and 2-hydroxy-oleic and linoleic acids are found inSalvia nilotica (Smith, 1971; Badami and Patil, 1981).

Fatty acids may also comprise epoxy groups. The most widely knownnatural epoxy fatty acid is vemolic acid(12,13-epoxy-octadec-cis-9-enoic acid) from the seed oils of Vernoniaspp and Euphorbia lagascae (Cuperus and Derksen, 1996). The epoxygenasegene of C. palaestina, which is related to but divergent from plantmembrane Δ12-oleate desaturases, has been functionally characterized andshown to use linoleate as a substrate (Lee et al. 1998).

In some organisms, conjugated fatty acids are produced by the activityof a conjugase (Crombie et al., 1984; Crombie et al., 1985; Fritsche etal., 1999; Cahoon et al., 2001; Qiu et al., 2001). The biosynthesis ofconjugated fatty acids such as calendulic acid, eleostearic acid orpunicic acid proceeds via the desaturation of oleic acid to linoleicacid by a Δ12-desaturase and a further desaturation in conjunction witha rearrangement of the Z9- or Z12-double bond to the conjutrienic fattyacid by a specific conjutriene-forming desaturase (conjugase).

There is a need for further methods of producing fatty acids inrecombinant cells and for more efficient production or production ofnovel fatty acids.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

SUMMARY OF THE INVENTION

The present inventors have identified novel enzymes having desaturase,conjugase, epoxygenase, and/or hydroxylase activity from insects of theOrder Coleoptera and Orthoptera.

Accordingly, the present invention provides a eukaryotic cell comprisingan exogenous nucleic acid encoding a polypeptide which is:

(i) a polypeptide comprising amino acids having a sequence as set forthin any one of SEQ ID NOs:16 to 30, 73 to 78, 80 and 134,

(ii) a polypeptide comprising amino acids having a sequence which is atleast 50% identical to any one or more of the sequences set forth in SEQID NOs: 16 to 30, 73 to 78, 80 and/or 134, and/or

(iii) a biologically active fragment of i) or ii),

wherein the polypeptide has one or more activities selected fromdesaturase, conjugase, epoxidase and hydroxylase activity.

In one embodiment, the polypeptide can be isolated from an insect of theOrder Coleoptera or Orthoptera. Examples of insect species from whichthe polypeptide can be isolated include, but are not limited to,Tribolium, Chauliognathus or Acheta.

In one embodiment, the polypeptide encoded by the exogenous nucleic acidis:

(i) a polypeptide comprising amino acids having a sequence as set forthin any one of SEQ ID NOs: 28, 73, and/or 134,

(ii) a polypeptide comprising amino acids having a sequence which is atleast 50% identical to any one or more of the sequences set forth in SEQID NOs: 28, 73, and/or 134, and/or

(iii) a biologically active fragment of (i) or (ii),

wherein the polypeptide has acyl-CoA Δ12 desaturase activity.

In another embodiment, the polypeptide is:

(i) a polypeptide comprising amino acids having a sequence as set forthin SEQ ID NO: 18 or SEQ ID NO: 19,

(ii) a polypeptide comprising amino acids having a sequence which is atleast 50% identical to a sequence set forth in SEQ ID NO: 18 or SEQ IDNO: 19, and/or

(iii) a biologically active fragment of (i) or (ii),

wherein the polypeptide has acyl-CoA Δ5 desaturase activity.

In yet another embodiment, the polypeptide is:

(i) a polypeptide comprising amino acids having a sequence as set forthin any one of SEQ ID NOs: 17, 22, 23, 27, 29, 74, 75 and/or 76,

(ii) a polypeptide comprising amino acids having a sequence which is atleast 50% identical to any one or more of the sequences set forth in SEQID NOs: 17, 22, 23, 27, 29, 74, 75 and/or 76, and/or

(iii) a biologically active fragment of (i) or (ii),

wherein the polypeptide has acyl-CoA Δ9 desaturase activity.

The present inventors are the first to identify a nucleic acid encodingan acyl-CoA Δ12 desaturase. Thus, the present invention provides aeukaryotic cell comprising an exogenous nucleic acid encoding anacyl-CoA Δ12 desaturase.

In one embodiment, the acyl-CoA Δ12 desaturase comprises:

(i) amino acids having a sequence as set forth in any one of SEQ ID NOs:28, 73, and/or 134,

(ii) amino acids having a sequence which is at least 50% identical toany one or more of the sequences set forth in SEQ ID NOs: 28, 73, and/or134, and/or

(iii) a biologically active fragment of (i) or (ii).

In one embodiment, the eukaryotic cell comprises an increased level of16:2^(Δ9,Δ12) and/or 18:2^(Δ9,Δ12) fatty acids relative to acorresponding eukaryotic cell lacking the exogenous nucleic acid.

In yet another embodiment, the eukaryotic cell comprises an increasedlevel of 16:2^(Δ9,Δ12) and/or 18:2^(Δ9,Δ12) fatty acids which areesterified to CoA.

The present inventors are also the first to identify an acyl-CoA Δ5desaturase which has a preference for a 18:0 and/or 16:0 fatty acidsubstrate when compared to a number of other substrates. Accordingly, ina further aspect the present invention provides a eukaryotic cellcomprising an exogenous nucleic acid encoding an acyl-CoA Δ5 desaturase;wherein the desaturase is more active on a 18:0 and/or 16:0 substratethan on a fatty acid substrate esterified to CoA, wherein the fatty acidis any one, two, three or all of a 18:1^(Δ9), 16:1^(Δ9), 20:0 and20:2^(Δ11Δ14).

In one embodiment, the acyl-CoA Δ5 desaturase comprises:

(i) amino acids having a sequence as set forth in SEQ ID NO: 18 or SEQID NO:19,

(ii) amino acids having a sequence which is at least 50% identical toSEQ ID NO: 18 or SEQ ID NO:19, and/or

(iii) a biologically active fragment of (i) or (ii).

In another embodiment, the eukaryotic cell comprises an increased levelof 16:1^(Δ5) and/or 18:1^(Δ5), fatty acids relative to a correspondingeukaryotic cell lacking the exogenous nucleic acid.

In yet another embodiment, the eukaryotic cell comprises an increasedlevel of 16:1^(Δ5) and/or 18:1^(Δ5) fatty acids which are esterified toCoA.

The present inventors are also the first to identify an acyl-CoA Δ9desaturase which is more active on a 14:0 substrate than on certainfatty acid substrates esterified to CoA. Thus, in a further aspect thepresent invention provides a eukaryotic cell comprising an exogenousnucleic acid encoding an acyl-CoA Δ9 desaturase, wherein the desaturaseis more active on a 14:0 substrate than on a fatty acid substrateesterified to CoA, wherein the fatty acid is 16:0 and/or 18:0.

In an embodiment, the acyl-CoA Δ9 desaturase comprises:

(i) amino acids having a sequence as set forth in SEQ ID NO: 23 or SEQID NO: 74,

(ii) amino acids having a sequence which is at least 50% identical toSEQ ID NO: 23 or SEQ ID NO:74, and/or

(iii) a biologically active fragment of (i) or (ii).

In one particular embodiment, the eukaryotic cell comprises an increasedlevel of 14:1^(Δ9) relative to a corresponding eukaryotic cell lackingthe exogenous nucleic acid.

In yet another embodiment, the eukaryotic cell comprises an increasedlevel of 14:1^(Δ9) which is esterified to CoA.

Preferably, the eukaryotic cell comprising the exogenous nucleic acid isa plant cell, a mammalian cell, an insect cell, a fungal cell or a yeastcell.

In one embodiment, the eukaryotic cell is in a plant or plant seed.

Preferably, the plant or plant seed is an oilseed plant or an oilseedrespectively.

In one aspect of the invention there is provided a process foridentifying a nucleic acid molecule involved in fatty acid modificationcomprising:

(i) obtaining a nucleic acid molecule operably linked to a promoter, thenucleic acid molecule encoding a polypeptide comprising amino acidshaving a sequence more closely related to SEQ ID NO: 28 than to SEQ IDNO: 135,

(ii) introducing the nucleic acid molecule into a cell or cell-freeexpression system in which the promoter is active,

(iii) determining whether the fatty acid composition is modifiedrelative to the cell or cell-free expression system before introductionof the nucleic acid molecule, and

(iv) optionally, selecting a nucleic acid molecule which modified thefatty acid composition.

In another aspect the present invention provides a process foridentifying a nucleic acid molecule involved in fatty acid modificationcomprising:

(i) obtaining a nucleic acid molecule operably linked to a promoter, thenucleic acid molecule encoding a polypeptide comprising amino acidshaving a sequence that is at least 50% identical to any one or more ofthe sequences set forth in SEQ ID NOs: 28, 73, and/or 134,

(ii) introducing the nucleic acid molecule into a cell or cell-freeexpression system in which the promoter is active,

(iii) determining whether the fatty acid composition is modifiedrelative to the cell or cell-free expression system before introductionof the nucleic acid molecule, and

(iv) optionally, selecting a nucleic acid molecule which modified thefatty acid composition.

In one embodiment, step (iv) comprises selecting a nucleic acid moleculeencoding an acyl-CoA Δ12 desaturase.

In another embodiment, the modified fatty acid composition comprises anincreased level of 16:2^(Δ9,Δ12) and/or 18:2^(Δ9,Δ12).

The present invention further provides a process for identifying anucleic acid molecule involved in fatty acid modification comprising:

(i) obtaining a nucleic acid molecule operably linked to a promoter, thenucleic acid molecule encoding a polypeptide comprising amino acidshaving a sequence that is at least 50% identical to any one or more ofthe sequences set forth in SEQ ID NOs: 18 and/or 19,

(ii) introducing the nucleic acid molecule into a cell or cell-freeexpression system in which the promoter is active,

(iii) determining whether the fatty acid composition is modifiedrelative to the cell or cell-free expression system before introductionof the nucleic acid, and

(iv) optionally, selecting a nucleic acid molecule which modified thefatty acid composition.

In one embodiment, step (iv) comprises selecting a nucleic acid moleculean acyl-CoA Δ5 desaturase, wherein the desaturase is more active on a18:0 and/or 16:0 substrate than on a fatty acid substrate esterified toCoA, wherein the fatty acid is any one, two, three or all of a18:1^(Δ9), 16:1^(Δ9), 20:0 and 20:2^(Δ11Δ14).

In another embodiment, the modified fatty acid composition comprises anincreased level of 16:1^(Δ5) and/or 18:1^(Δ5) fatty acids.

The present invention further provides a process for identifying anucleic acid molecule involved in fatty acid modification comprising:

(i) obtaining a nucleic acid molecule operably linked to a promoter, thenucleic acid molecule encoding a polypeptide comprising amino acidshaving a sequence that is at least 50% identical to any one or more ofthe sequences set forth in SEQ ID NOs: 17, 22, 23, 27, 29, 74, 75 and/or76,

(ii) introducing the nucleic acid molecule into a cell or cell-freeexpression system in which the promoter is active,

(iii) determining whether the fatty acid composition is modifiedrelative to the cell or cell-free expression system before introductionof the nucleic acid, and

(iv) optionally, selecting a nucleic acid molecule which modified thefatty acid composition.

In one embodiment, step (iv) comprises selecting a nucleic acid moleculeencoding an acyl-CoA Δ9 desaturase, wherein the desaturase is moreactive on a 14:0 substrate than on a fatty acid substrate esterified toCoA, wherein the fatty acid is 16:0 and/or 18:0.

In another embodiment, the modified fatty acid composition comprises anincreased level of 14:1^(Δ9).

In yet another embodiment, the polypeptide encoded by the nucleic acidmolecule is an insect polypeptide or mutant thereof.

The present invention further provides a substantially purified orrecombinant polypeptide which is an acyl-CoA Δ12 desaturase.

In one embodiment, the polypeptide is:

(i) a polypeptide comprising amino acids having a sequence as set forthin any one of SEQ ID NOs: 28, 73, and/or 134,

(ii) a polypeptide comprising amino acids having a sequence which is atleast 50% identical to any one or more of the sequences set forth in SEQID NOs: 28, 73, and/or 134, and/or

(iii) a biologically active fragment of (i) or (ii).

The present invention further provides a substantially purified orrecombinant polypeptide which is an acyl-CoA Δ5 desaturase, wherein thedesaturase is more active on a 18:0 and/or 16:0 substrate than on afatty acid substrate esterified to CoA, wherein the fatty acid is anyone, two, three or all of a 18:1^(Δ9), 16:1^(Δ9), 20:0 and20:2^(Δ11Δ14).

In one embodiment, the polypeptide is:

(i) a polypeptide comprising amino acids having a sequence as set forthin SEQ ID NO: 18 or SEQ ID NO:19,

(ii) a polypeptide comprising amino acids having a sequence which is atleast 50% identical to SEQ ID NO:18 and/or SEQ ID NO:19, and/or

(iii) a biologically active fragment of (i) or (ii).

The present invention further provides a substantially purified orrecombinant polypeptide which is an acyl-CoA Δ9 desaturase, wherein thedesaturase is more active on a 14:0 substrate than on a fatty acidsubstrate esterified to CoA, wherein the fatty acid is 16:0 and/or 18:0.

In one embodiment, the polypeptide is:

(i) a polypeptide comprising amino acids having a sequence as set forthin SEQ ID NO: 17 or SEQ ID NO:74,

(ii) a polypeptide comprising amino acids having a sequence which is atleast 50% identical to one or more of the sequences as set forth in SEQID NO: 17 and/or SEQ ID NO: 74, and/or

(iii) a biologically active fragment of (i) or (ii).

The present invention further provides a substantially purified orrecombinant polypeptide which is:

(i) a polypeptide comprising amino acids having a sequence as set forthin any one of SEQ ID NOs:16 to 30, 73 to 78, 80 and 134,

(ii) a polypeptide comprising amino acids having a sequence which is atleast 50% identical to any one or more of the sequences set forth in SEQID NOs: 16 to 30, 73 to 78, 80 and/or 134, and/or

(iii) a biologically active fragment of i) or ii),

wherein the polypeptide has one or more an activities selected fromdesaturase, conjugase, epoxidase and/or hydroxylase activity.

Preferably, the polypeptide comprises amino acids having a sequencewhich is at least 90% identical to any one or more of the sequences setforth in SEQ ID NOs: 16 to 30, 73 to 78, 80 and/or 134.

In one embodiment, the polypeptide can be isolated from an insect of theOrder Coleoptera or Orthoptera.

Preferably, the polypeptide has desaturase activity upon a carbon-carbonbond located at any one of the Δ2 to Δ15 positions of a fatty acid.

In one embodiment, the polypeptide has desaturase activity on a C16 orC18 fatty acid.

In another embodiment, the polypeptide is a fusion protein furthercomprising at least one other polypeptide sequence.

The present invention further provides an isolated and/or exogenouspolynucleotide comprising:

(i) a sequence of nucleotides selected from any one of SEQ ID NOs: 1 to15, 67 to 72, 79 and 133,

(ii) a sequence of nucleotides encoding a polypeptide of the invention,

(iii) a sequence of nucleotides which are at least 50% identical to oneor more of the sequences set forth in SEQ ID NOs: 1 to 15, 67 to 72, 79and/or 133, and/or

(iv) a sequence which hybridises to any one of (i) to (iii) understringent conditions.

Also provided is a vector comprising a polynucleotide of the invention.

In one embodiment, the polynucleotide is operably linked to a promoter.

The present invention further provides a cell comprising the recombinantpolypeptide according to the invention, the exogenous polynucleotide ofthe invention and/or the vector of the invention.

Preferably, the cell is a plant, fungal, yeast, bacterial or animalcell. More preferably, the cell is a eukaryote cell.

The present invention further provides a method of producing thepolypeptide according to the invention, the method comprising expressingin a cell or cell free expression system the polynucleotide of theinvention.

In one embodiment, the method further comprises isolating thepolypeptide.

The present invention further provides a transgenic non-human organismcomprising a cell according to the invention.

In one embodiment, the organism is a transgenic plant.

The present invention further provides a seed comprising the cellaccording to the invention.

The present invention further provides oil produced by, or obtainedfrom, the cell according to the invention, the transgenic non-humanorganism of the invention, or the seed of the invention.

In one embodiment, the oil comprises fatty acids 16:0, 16:1^(Δ5), 18:0and 18:1^(Δ5), wherein the ratio of the total amount of 18:1^(Δ5) to18:0 in the oil is between 100:1 and 1:2, and wherein the fatty acid ofthe oil comprises less than 10%, or less than 5% (w/w) 20:1^(Δ5).

In another embodiment, at least 43%, or at least 50%, or at least 60%,of the C18 fatty acid of the oil is 18:1^(Δ5).

In yet another embodiment, the fatty acid of the oil comprises at least3.0% (w/w), or at least 5% (w/w) or at least 10% (w/w), 18:1^(Δ5) as apercentage of the total fatty acid of the oil.

In another embodiment, the oil comprises less than 10% (w/w), or lessthan 5% (w/w), 18:3^(Δ9,Δ12,Δ15) (ALA) as a percentage of the totalfatty acid in the oil.

In one embodiment, the oil is obtained by extraction of oil from anoilseed.

The present invention further provides an oil comprising fatty acids16:0, 16:1^(Δ5), 18:0 and 18:1^(Δ5), wherein the ratio of the totalamount of 18:1^(Δ5) to 18:0 in the oil is between 100:1 and 1:2, andwherein the fatty acid of the oil comprises less than 10% (w/w), or lessthan 5%, 20:1^(Δ5).

The present invention further provides an oil comprising fatty acids,wherein at least 43%, or at least 50%, or at least 60%, of the C18 fattyacid of the oil is 18:1^(Δ5).

The invention further provides an oil comprising fatty acids comprisingat least 3.0% (w/w), or at least 5% (w/w) or at least 10% (w/w),18:1^(Δ5) as a percentage of the total fatty acid of the oil.

In one embodiment, oil comprises less than 10% (w/w), or less than 5%(w/w), 18:3^(Δ9,Δ12,Δ15) (ALA) as a percentage of the total fatty acidin the oil.

The present invention further provides a fatty acid produced by, orobtained from, the cell according to the invention, the transgenicnon-human organism of the invention, or the seed of the invention.

The invention also provides a method of producing oil containingunsaturated fatty acids, the method comprising extracting oil from thecell according to the invention, the transgenic non-human organism ofthe invention, or the seed of the invention.

The present invention further provides a composition comprising a cellaccording to the invention, the polypeptide according to the invention,a polynucleotide according to the invention, a vector of the invention,an oil according to the invention or a fatty acid of the invention.

The invention also provides feedstuffs, cosmetics or chemicalscomprising the oil according to the invention or a fatty acid of theinvention.

The present invention also provides a method of performing a desaturasereaction, the method comprising contacting a substrate saturated,monounsaturated or polyunsaturated fatty acid esterified to CoA with thepolypeptide of the invention

The present invention further provides a substantially purifiedantibody, or fragment thereof, that specifically binds a polypeptide ofthe invention

The invention further provides a method of treating or preventing acondition which would benefit from a PUFA, the method comprisingadministering to a subject a cell according to the invention, thepolypeptide according to the invention, a polynucleotide according tothe invention, a vector of the invention, an oil according to theinvention, a fatty acid of the invention and/or a feedstuff of theinvention.

In one embodiment, the condition is cardiac arrhythmia's, angioplasty,inflammation, asthma, psoriasis, osteoporosis, kidney stones, AIDS,multiple sclerosis, rheumatoid arthritis, Crohn's disease,schizophrenia, cancer, foetal alcohol syndrome, attention deficienthyperactivity disorder, cystic fibrosis, phenylketonuria, unipolardepression, aggressive hostility, adrenoleukodystophy, coronary heartdisease, hypertension, diabetes, obesity, Alzheimer's disease, chronicobstructive pulmonary disease, ulcerative colitis, restenosis afterangioplasty, eczema, high blood pressure, platelet aggregation,gastrointestinal bleeding, endometriosis, premenstrual syndrome, myalgicencephalomyelitis, chronic fatigue after viral infections or an oculardisease.

The present invention also provides use of a cell according to theinvention, the polypeptide according to the invention, a polynucleotideaccording to the invention, a vector of the invention, an oil accordingthe invention or a fatty acid of the invention and/or a feedstuff of theinvention for the manufacture of a medicament for treating or preventinga condition which would benefit from a PUFA.

As will be apparent, preferred features and characteristics of oneaspect of the invention are applicable to many other aspects of theinvention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1. A comparison of the nucleic acid sequence obtained from the geneprediction program and that of the authentic RT-PCR product forTribdesat 2b. Dots indicate sequence identity, while differentnucleotides are indicated.

FIG. 2. A comparison of the nucleic acid sequence obtained from the geneprediction program and that of the authentic RT-PCR product forTribdesat 3. Dots indicate sequence identity, while differentnucleotides are indicated.

FIG. 3. A comparison of the nucleic acid sequence obtained from the geneprediction program and that of the authentic RT-PCR product forTribdesat 6b. Dots indicate sequence identity, while differentnucleotides are indicated.

FIG. 4. A comparison of the nucleic acid sequence obtained from the geneprediction program and that of the authentic RT-PCR product forTribdesat 10. Dots indicate sequence identity, while differentnucleotides are indicated.

FIG. 5. A comparison of the nucleic acid sequence obtained from the geneprediction program and that of the authentic RT-PCR product forTribdesat 11. Dots indicate sequence identity, while differentnucleotides are indicated.

FIG. 6. GC/MS trace and spectrum showing linoleic acid (18:2 Δ9,Δ12)production by Tribdesat 10 in Arabidopsis, with Arabidopsis Fad1/Fae2control.

FIG. 7. A. Gas chromatography (GC) of yeast fatty acid methyl estersfrom S. cerivisiae expressing AdD12Des. B. Gas chromatography (GC) ofyeast fatty acid methyl esters from S. cerivisiae, vector only. C.Confirmation of the double bond positions of C16:2 and C18:2 products byGC-mass spectrometry.

FIG. 8. GC analysis of yeast ole1 cells expressing pYES2 vector only(Panel A) or A. domesticus Δ12-desaturase in pXZP282 (Panel B) after fedwith mixture of C14:0 and C15:0.

FIG. 9. Phylogenetic analysis of representative acyl-CoA or acyl-lipiddesaturase protein sequences.

FIG. 10. Phylogenetic analysis of representative acyl-CoA or acyl-lipiddesaturase protein sequences.

KEY TO THE SEQUENCE LISTING

SEQ ID NO: 1—coding sequence of Tribolium desaturase 1SEQ ID NO: 2—coding sequence of Tribolium desaturase 2aSEQ ID NO: 3—coding sequence of Tribolium desaturase 2bSEQ ID NO: 4—coding sequence of Tribolium desaturase 2cSEQ ID NO: 5—coding sequence of Tribolium desaturase 3SEQ ID NO: 6—coding sequence of Tribolium desaturase 4SEQ ID NO: 7—coding sequence of Tribolium desaturase 5SEQ ID NO: 8—coding sequence of Tribolium desaturase 6aSEQ ID NO: 9—coding sequence of Tribolium desaturase 6bSEQ ID NO: 10—coding sequence of Tribolium desaturase 7aSEQ ID NO: 11—coding sequence of Tribolium desaturase 7bSEQ ID NO: 12—coding sequence of Tribolium desaturase 8SEQ ID NO: 13—coding sequence of Tribolium desaturase 10SEQ ID NO: 14—coding sequence of Tribolium desaturase 11SEQ ID NO: 15—coding sequence of Tribolium desaturase 12SEQ ID NO: 16—amino acid sequence of Tribolium desaturase 1SEQ ID NO: 17—amino acid sequence of Tribolium desaturase 2aSEQ ID NO: 18—amino acid sequence of Tribolium desaturase 2bSEQ ID NO: 19—amino acid sequence of Tribolium desaturase 2cSEQ ID NO: 20—amino acid sequence of Tribolium desaturase 3SEQ ID NO: 21—amino acid sequence of Tribolium desaturase 4SEQ ID NO: 22—amino acid sequence of Tribolium desaturase 5SEQ ID NO: 23—amino acid sequence of Tribolium desaturase 6aSEQ ID NO: 24—amino acid sequence of Tribolium desaturase 6bSEQ ID NO: 25—amino acid sequence of Tribolium desaturase 7aSEQ ID NO: 26—amino acid sequence of Tribolium desaturase 7bSEQ ID NO: 27—amino acid sequence of Tribolium desaturase 8SEQ ID NO: 28—amino acid sequence of Tribolium desaturase 10SEQ ID NO: 29—amino acid sequence of Tribolium desaturase 11SEQ ID NO: 30—amino acid sequence of Tribolium desaturase 12SEQ ID NO: 31—conserved insect desaturase sequenceSEQ ID NOs: 32 to 66—oligonucleotide primersSEQ ID NO: 67—coding sequence of Chauliognathus desaturase CL1SEQ ID NO: 68—coding sequence of Chauliognathus desaturase CL3SEQ ID NO: 69—partial coding sequence of Chauliognathus desaturase CL6SEQ ID NO: 70—partial coding sequence of Chauliognathus desaturase CL7SEQ ID NO: 71—partial coding sequence of Chauliognathus desaturase CL8SEQ ID NO: 72—partial coding sequence of Chauliognathus desaturase CL9SEQ ID NO: 73—amino acid sequence of Chauliognathus desaturase CL1SEQ ID NO: 74—amino acid sequence of Chauliognathus desaturase CL3SEQ ID NO: 75—partial amino acid sequence of Chauliognathus desaturaseCL6SEQ ID NO: 76—partial amino acid sequence of Chauliognathus desaturaseCL7SEQ ID NO: 77—partial amino acid sequence of Chauliognathus desaturaseCL8SEQ ID NO: 78—partial amino acid sequence of Chauliognathus desaturaseCL9SEQ ID NO: 79—coding sequence of Chauliognathus desaturase CN1SEQ ID NO: 80—amino acid sequence of Chauliognathus desaturase CN1SEQ ID NOs: 81 to 103—oligonucleotide primersSEQ ID NOs: 104 to 111—conserved desaturase motifsSEQ ID NOs: 112 to 122—histidine boxes in desaturase sequencesSEQ ID NOs: 123 to 126—signature motifsSEQ ID NOs: 127 to 130—oligonucleotidesSEQ ID NOs: 131 and 132—conserved regionsSEQ ID NO: 133—coding sequence of Acheta domesticus desaturase AdD12DesSEQ ID NO: 134—amino acid sequence of Acheta domesticus desaturaseAdD12DesSEQ ID NO: 135—Arabidopsis thaliana FAD2 Δ12 desaturase

DETAILED DESCRIPTION OF THE INVENTION General Techniques

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, immunology, immunohistochemistry, protein chemistry,and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present), and are incorporated herein by reference.

Selected Definitions

As used herein, the term “fatty acid” refers to a carboxylic acid (ororganic acid), often with a long aliphatic tail, either saturated orunsaturated. Typically fatty acids have a carbon-carbon bonded chain ofat least 8 carbon atoms in length, more preferably at least 12 carbonsin length. Most naturally occurring fatty acids have an even number ofcarbon atoms because their biosynthesis involves acetate which has twocarbon atoms. The fatty acids may be in a free state (non-esterified) orin an esterified form such as part of a triglyceride, diacylglyceride,monoacylglyceride, acyl-CoA (thio-ester) bound or other bound form. Thefatty acid may be esterified as a phospholipid such as aphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerolforms.

“Saturated fatty acids” do not contain any double bonds or otherfunctional groups along the chain. The term “saturated” refers tohydrogen, in that all carbons (apart from the carboxylic acid [—COOH]group) contain as many hydrogens as possible. In other words, the omega(ω) end contains 3 hydrogens (CH3-) and each carbon within the chaincontains 2 hydrogens (—CH2-).

“Unsaturated fatty acids” are of similar form to saturated fatty acids,except that one or more alkene functional groups exist along the chain,with each alkene substituting a singly-bonded “—CH2-CH2-” part of thechain with a doubly-bonded “—CH═CH—” portion (that is, a carbon doublebonded to another carbon). The two next carbon atoms in the chain thatare bound to either side of the double bond can occur in a cis or transconfiguration.

As used herein, the terms “monounsaturated fatty acid” refers to a fattyacid which comprises at least 12 carbon atoms in its carbon chain andonly one alkene group in the chain. As used herein, the terms“polyunsaturated fatty acid” or “PUFA” refer to a fatty acid whichcomprises at least 12 carbon atoms in its carbon chain and at least twoalkene groups (carbon-carbon double bonds). Ordinarily, the number ofcarbon atoms in the carbon chain of the fatty acids refers to anunbranched carbon chain. If the carbon chain is branched, the number ofcarbon atoms excludes those in sidegroups. In one embodiment, thelong-chain polyunsaturated fatty acid is an ω3 fatty acid, that is,having a desaturation (carbon-carbon double bond) in the thirdcarbon-carbon bond from the methyl end of the fatty acid. In anotherembodiment, the long-chain polyunsaturated fatty acid is an ω6 fattyacid, that is, having a desaturation (carbon-carbon double bond) in thesixth carbon-carbon bond from the methyl end of the fatty acid.

As used herein, the terms “long-chain polyunsaturated fatty acid” or“LC-PUFA” refer to a fatty acid which comprises at least 20 carbon atomsin its carbon chain and at least two carbon-carbon double bonds.

As used herein, the term “desaturase” refers to an enzyme which iscapable of introducing a carbon-carbon double bond into the acyl groupof a fatty acid substrate, which is typically in an esterified form suchas, for example, fatty acid CoA esters. The acyl group may be esterifiedto a phospholipid such as phosphatidyl choline, or to acyl carrierprotein (ACP), or in a preferred embodiment to CoA. Desaturasesgenerally may be categorized into three groups accordingly.

As used herein, the term “Δ12 desaturase” refers to a protein whichperforms a desaturase reaction that introduces a carbon-carbon doublebond located at the 12^(th) bond from the carboxyl end and has greateractivity in desaturation at this position than any other position. Inone embodiment, the Δ12 desaturase activity includes oleoyl-CoA Δ12desaturase activity. In another embodiment, the Δ12 desaturase activityincludes palmitoleoyl-CoA Δ12 desaturase activity. These fatty acids maybe in an esterified form, such as, for example, as part of aphospholipid. Examples of Δ12 desaturases include proteins comprising anamino acid sequence provided in SEQ ID NOs: 28, 78 and 134.

There are two types of Δ12 desaturases; acyl-CoA Δ12 desaturases andacyl-PC Δ12 desaturases, which are predominantly active on acyl-CoA andacyl-PC linked 18:1 substrates, respectively. However, Δ9 desaturasesmay also be active on an acyl-ACP (acyl carrier protein).

As used herein, the term “acyl-CoA Δ12 desaturase activity” or “acyl-CoAΔ12 desaturase” refers to the desaturase having greater activity on anacyl-CoA substrate than an acyl-lipid (such as acyl-PC) and/or acyl-ACPsubstrate. In an embodiment, the activity is at least two-fold greater.

As used herein, a “Δ5 desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon bond located at the5^(th) bond from the carboxyl end and has greater activity indesaturation at this position than any other position. In one embodimentthe Δ5 desaturase has acyl-CoA-stearoyl Δ5 desaturase activity. Inanother embodiment, the Δ5 desaturase has acyl-CoA-palmitoyl Δ5desaturase activity. In one embodiment, the enzyme Δ5 desaturasecatalyses the desaturation of C20 LC-PUFA, converting dihomo-γ-linoleicacid DGLA to arachidonic acid (ARA, 20:40ω6) and ETA to EPA (20:50ω3).

As used herein, the term “acyl-CoA Δ5 desaturase activity” or “acyl-CoAΔ5 desaturase” refers to the desaturase having greater activity on anacyl-CoA substrate than an acyl-lipid (such as acyl-PC) and/or acyl-ACPsubstrate. In an embodiment, the activity is at least two-fold greater.

As used herein, a “Δ9 desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon bond located at the9^(th) bond from the carboxyl end and has greater activity indesaturation at this position than any other position. Examples of Δ9desaturase activity include myristoyl-CoA Δ9 desaturase activity,stearoyl-CoA Δ9 desaturase activity, palmitoyl-CoA Δ9 desaturaseactivity, lignoceroyl-CoA Δ9 desaturase activity and behenoyl-CoA Δ9desaturase activity.

As used herein, the term “acyl-CoA Δ9 desaturase activity” or “acyl-CoAΔ9 desaturase” refers to the desaturase having greater activity on anacyl-CoA substrate than an acyl-lipid (such as acyl-PC) and/or acyl-ACPsubstrate. In an embodiment, the activity is at least two-fold greater.

As used herein, the term “conjugase” refers to a conjutriene-formingdesaturase.

The term “epoxidase” as used herein refers to an enzyme that introducesan epoxy group into a fatty acid resulting in the production of an epoxyfatty acid. In preferred embodiment, the epoxy group is introduced atthe 2nd and/or 12th carbon on a fatty acid chain, especially of a C16 orC18 fatty acid chain.

“Hydroxylase”, as used herein, refers to an enzyme that introduces ahydroxyl group into a fatty acid resulting in the production of ahydroxylated fatty acid. In a preferred embodiment, the hydroxyl groupis introduced at the 2nd, 12th and/or 17th carbon on a C18 fatty acidchain. In another preferred embodiment, the hydroxyl group is introducedat the 15th carbon on a C16 fatty acid chain.

The term “plant” includes whole plants, vegetative structures (forexample, leaves, stems), roots, floral organs/structures, seed(including embryo, endosperm, and seed coat), plant tissue (for example,vascular tissue, ground tissue, and the like), cells and progeny of thesame.

A “transgenic plant”, “genetically modified plant” or variations thereofrefers to a plant that contains a gene construct (“transgene”) not foundin a wild-type plant of the same species, variety or cultivar. A“transgene” as referred to herein has the normal meaning in the art ofbiotechnology and includes a genetic sequence which has been produced oraltered by recombinant DNA or RNA technology and which has beenintroduced into the plant cell. The transgene may include geneticsequences derived from a plant cell. Typically, the transgene has beenintroduced into the plant by human manipulation such as, for example, bytransformation but any method can be used as one of skill in the artrecognizes.

The terms “seed” and “grain” are used interchangeably herein. “Grain”generally refers to mature, harvested grain but can also refer to grainafter imbibition or germination, according to the context. Mature graincommonly has a moisture content of less than about 18-20%.

“Operably linked” as used herein refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of transcriptional regulatoryelement (promoter) to a transcribed sequence. For example, a promoter isoperably linked to a coding sequence, such as a polynucleotide definedherein, if it stimulates or modulates the transcription of the codingsequence in an appropriate cell. Generally, promoter transcriptionalregulatory elements that are operably linked to a transcribed sequenceare physically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory elements, such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

As used herein, the term “gene” is to be taken in its broadest contextand includes the deoxyribonucleotide sequences comprising the proteincoding region of a structural gene and including sequences locatedadjacent to the coding region on both the 5′ and 3′ ends for a distanceof at least about 2 kb on either end and which are involved inexpression of the gene. The sequences which are located 5′ of the codingregion and which are present on the mRNA are referred to as 5′non-translated sequences. The sequences which are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region which may be interrupted with non-codingsequences termed “introns” or “intervening regions” or “interveningsequences.” Introns are segments of a gene which are transcribed intonuclear RNA (hnRNA); introns may contain regulatory elements such asenhancers. Introns are removed or “spliced out” from the nuclear orprimary transcript; introns therefore are absent in the messenger RNA(mRNA) transcript. The mRNA functions during translation to specify thesequence or order of amino acids in a nascent polypeptide. The term“gene” includes a synthetic or fusion molecule encoding all or part ofthe proteins of the invention described herein and a complementarynucleotide sequence to any one of the above.

As used herein, the term “can be isolated from” means that thepolynucleotide or encoded polypeptide is naturally produced by anorganism, particularly an insect.

Polypeptides/Peptides

By “substantially purified polypeptide” or “purified polypeptide” wemean a polypeptide that has generally been separated from the lipids,nucleic acids, other peptides, and other contaminating molecules withwhich it is associated in its native state. Preferably, thesubstantially purified polypeptide is at least 60% free, more preferablyat least 75% free, and more preferably at least 90% free from othercomponents with which it is naturally associated.

The term “recombinant” in the context of a polypeptide refers to thepolypeptide when produced by a cell, or in a cell-free expressionsystem, in an altered amount or at an altered rate compared to itsnative state. In one embodiment the cell is a cell that does notnaturally produce the polypeptide. However, the cell may be a cell whichcomprises a non-endogenous gene that causes an altered amount of thepolypeptide to be produced. A recombinant polypeptide of the inventionincludes polypeptides which have not been separated from othercomponents of the transgenic (recombinant) cell, or cell-free expressionsystem, in which it is produced, and polypeptides produced in such cellsor cell-free systems which are subsequently purified away from at leastsome other components.

The terms “polypeptide” and “protein” are generally usedinterchangeably.

The % identity of a polypeptide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. The query sequence is at least 15 aminoacids in length, and the GAP analysis aligns the two sequences over aregion of at least 15 amino acids. More preferably, the query sequenceis at least 50 amino acids in length, and the GAP analysis aligns thetwo sequences over a region of at least 50 amino acids. More preferably,the query sequence is at least 100 amino acids in length and the GAPanalysis aligns the two sequences over a region of at least 100 aminoacids. Even more preferably, the query sequence is at least 250 aminoacids in length and the GAP analysis aligns the two sequences over aregion of at least 250 amino acids. Even more preferably, the GAPanalysis aligns two sequences over their entire length.

As used herein a “biologically active” fragment is a portion of apolypeptide of the invention which maintains a defined activity of thefull-length polypeptide, namely possessing desaturase, conjugase,epoxidase, and/or hydroxylase activity. Biologically active fragmentscan be any size as long as they maintain the defined activity.Preferably, the biologically active fragment maintains at least 10% ofthe activity of the full length protein.

With regard to a defined polypeptide/enzyme, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide/enzyme comprisesan amino acid sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 76%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, more preferably at least 93%, morepreferably at least 94%, more preferably at least 95%, more preferablyat least 96%, more preferably at least 97%, more preferably at least98%, more preferably at least 99%, more preferably at least 99.1%, morepreferably at least 99.2%, more preferably at least 99.3%, morepreferably at least 99.4%, more preferably at least 99.5%, morepreferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ ID NO.

With regard to the acyl-CoA Δ5 desaturases of the invention, this is thefirst demonstration of an animal desaturase that can produce5-hexadecenoic and 5-octadecenoic acids and uses an acyl-CoA fatty acidas a substrate.

With regard to the acyl-CoA Δ12 desaturases of the invention, this isthe first known demonstration of Δ12 desaturase activity by an enzymenaturally produced by an animal. Furthermore, this is the firstdemonstration of a desaturase that can produce linoleic acid and uses anacyl-CoA fatty acid as a substrate.

Amino acid sequence mutants of the polypeptides of the present inventioncan be prepared by introducing appropriate nucleotide changes into anucleic acid of the present invention, or by in vitro synthesis of thedesired polypeptide. Such mutants include, for example, deletions,insertions or substitutions of residues within the amino acid sequence.A combination of deletion, insertion and substitution can be made toarrive at the final construct, provided that the final peptide productpossesses the desired characteristics.

Mutant (altered) peptides can be prepared using any technique known inthe art. For example, a polynucleotide of the invention can be subjectedto in vitro mutagenesis. Such in vitro mutagenesis techniques includesub-cloning the polynucleotide into a suitable vector, transforming thevector into a “mutator” strain such as the E. coli XL-1 red (Stratagene)and propagating the transformed bacteria for a suitable number ofgenerations. In another example, the polynucleotides of the inventionare subjected to DNA shuffling techniques as broadly described byHarayama (1998). Products derived from mutated/altered DNA can readilybe screened using techniques described herein to determine if theypossess desaturase, conjugase, epoxidase, and/or hydroxylase activity.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries, e.g., by (1) substituting first with conservative amino acidchoices and then with more radical selections depending upon the resultsachieved, (2) deleting the target residue, or (3) inserting otherresidues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. The sites of greatest interest for substitutional mutagenesisinclude sites identified as the active site(s). Other sites of interestare those in which particular residues obtained from various strains orspecies are identical. These positions may be important for biologicalactivity. These sites, especially those falling within a sequence of atleast three other identically conserved sites, are preferablysubstituted in a relatively conservative manner. Such conservativesubstitutions are shown in Table 1 under the heading of “exemplarysubstitutions”.

TABLE 1 Exemplary substitutions. Original Exemplary ResidueSubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,ala

In a preferred embodiment a mutant/variant polypeptide has one or two orthree or four conservative amino acid changes when compared to anaturally occurring polypeptide. Details of conservative amino acidchanges are provided in Table 1. In a preferred embodiment, the changesare not in one or more of the following motifs: AGAHRLW, SETDAD,FFFSHVG, QKKY, NSAAH, GEGWHNYHH, PWDY, and GWAY. In a particularlypreferred embodiment, the changes are not in each of the followingmotifs: AGAHRLW, SETDAD, FFFSHVG, QKKY, NSAAH, GEGWHNYHH, PWDY, andGWAY. As the skilled person would be aware, such minor changes canreasonably be predicted not to alter the activity of the polypeptidewhen expressed in a recombinant cell.

Furthermore, if desired, unnatural amino acids or chemical amino acidanalogues can be introduced as a substitution or addition into thepolypeptides of the present invention. Such amino acids include, but arenot limited to, the D-isomers of the common amino acids,2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid,2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as n-methylamino acids, Cα-methyl amino acids, Nα-methyl amino acids, and aminoacid analogues in general.

Also included within the scope of the invention are polypeptides of thepresent invention which are differentially modified during or aftersynthesis, e.g., by biotinylation, benzylation, glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. These modifications may serve toincrease the stability and/or bioactivity of the polypeptide of theinvention.

Polypeptides of the present invention can be produced in a variety ofways, including production and recovery of natural polypeptides,production and recovery of recombinant polypeptides, and chemicalsynthesis of the polypeptides. In one embodiment, an isolatedpolypeptide of the present invention is produced by culturing a cellcapable of expressing the polypeptide under conditions effective toproduce the polypeptide, and recovering the polypeptide. A preferredcell to culture is a recombinant cell of the present invention.Effective culture conditions include, but are not limited to, effectivemedia, bioreactor, temperature, pH and oxygen conditions that permitpolypeptide production. An effective medium refers to any medium inwhich a cell is cultured to produce a polypeptide of the presentinvention. Such medium typically comprises an aqueous medium havingassimilable carbon, nitrogen and phosphate sources, and appropriatesalts, minerals, metals and other nutrients, such as vitamins. Cells ofthe present invention can be cultured in conventional fermentationbioreactors, shake flasks, test tubes, microtiter dishes, and petriplates. Culturing can be carried out at a temperature, pH and oxygencontent appropriate for a recombinant cell. Such culturing conditionsare within the expertise of one of ordinary skill in the art.

Polynucleotides

By “isolated polynucleotide” we mean a polynucleotide which hasgenerally been separated from the polynucleotide sequences with which itis associated or linked in its native state. Preferably, the isolatedpolynucleotide is at least 60% free, more preferably at least 75% free,and more preferably at least 90% free from other components with whichit is naturally associated. Furthermore, the term “polynucleotide” isused interchangeably herein with the terms “nucleic acid molecule”,“gene” and “mRNA”.

The term “exogenous” in the context of a polynucleotide refers to thepolynucleotide when present in a cell, or in a cell-free expressionsystem, in an altered amount compared to its native state. In oneembodiment, the cell is a cell that does not naturally comprise thepolynucleotide. However, the cell may be a cell which comprises anon-endogenous polynucleotide resulting in an altered amount ofproduction of the encoded polypeptide. An exogenous polynucleotide ofthe invention includes polynucleotides which have not been separatedfrom other components of the transgenic (recombinant) cell, or cell-freeexpression system, in which it is present, and polynucleotides producedin such cells or cell-free systems which are subsequently purified awayfrom at least some other components. The exogenous polynucleotide(nucleic acid) can be a contiguous stretch of nucleotides existing innature, or comprise two or more contiguous stretches of nucleotides fromdifferent sources (naturally occurring and/or synthetic) joined to forma single polynucleotide. Typically such chimeric polynucleotidescomprise at least an open reading frame encoding a polypeptide of theinvention operably linked to a promoter suitable of drivingtranscription of the open reading frame in a cell of interest.

“Polynucleotide” refers to a oligonucleotide, polynucleotide or anyfragment thereof. It may be DNA or RNA of genomic or synthetic origin,double-stranded or single-stranded, and combined with carbohydrate,lipids, protein, or other materials to perform a particular activitydefined herein.

The % identity of a polynucleotide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. The query sequence is at least 45nucleotides in length, and the GAP analysis aligns the two sequencesover a region of at least 45 nucleotides. Preferably, the query sequenceis at least 150 nucleotides in length, and the GAP analysis aligns thetwo sequences over a region of at least 150 nucleotides. Even morepreferably, the query sequence is at least 300 nucleotides in length andthe GAP analysis aligns the two sequences over a region of at least 300nucleotides. Even more preferably, the GAP analysis aligns two sequencesover their entire length.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, more preferably at least 93%, more preferably at least 94%, morepreferably at least 95%, more preferably at least 96%, more preferablyat least 97%, more preferably at least 98%, more preferably at least99%, more preferably at least 99.1%, more preferably at least 99.2%,more preferably at least 99.3%, more preferably at least 99.4%, morepreferably at least 99.5%, more preferably at least 99.6%, morepreferably at least 99.7%, more preferably at least 99.8%, and even morepreferably at least 99.9% identical to the relevant nominated SEQ ID NO.

A polynucleotide of the present invention may selectively hybridise to apolynucleotide that encodes a polypeptide of the present invention understringent conditions. As used herein, under stringent conditions arethose that (1) employ low ionic strength and high temperature forwashing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodSO₄at 50° C.; (2) employ during hybridisation a denaturing agent such asformamide, for example, 50% (vol/vol) formamide with 0.1% bovine serumalbumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphatebuffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or(3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate),50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextransulfate at 42° C. in 0.2×SSC and 0.1% SDS.

Polynucleotides of the present invention may possess, when compared tonaturally occurring molecules, one or more mutations which aredeletions, insertions, or substitutions of nucleotide residues. Mutantscan be either naturally occurring (that is to say, isolated from anatural source) or synthetic (for example, by performing site-directedmutagenesis or DNA shuffling on the nucleic acid as described above). Itis thus apparent that polynucleotides of the invention can be eithernaturally occurring or recombinant.

Oligonucleotides of the present invention can be RNA, DNA, orderivatives of either. The minimum size of such oligonucleotides is thesize required for the formation of a stable hybrid between anoligonucleotide and a complementary sequence on a polynucleotide of thepresent invention. Preferably, the oligonucleotides are at least 15nucleotides, more preferably at least 18 nucleotides, more preferably atleast 19 nucleotides, more preferably at least 20 nucleotides, even morepreferably at least 25 nucleotides in length. The present inventionincludes oligonucleotides that can be used as, for example, probes toidentify nucleic acid molecules, or primers to produce nucleic acidmolecules. Oligonucleotides of the present invention used as a probe aretypically conjugated with a label such as a radioisotope, an enzyme,biotin, a fluorescent molecule or a chemiluminescent molecule.

Recombinant Vectors

One embodiment of the present invention includes a recombinant vector,which comprises at least one isolated polynucleotide molecule of thepresent invention, inserted into any vector capable of delivering thepolynucleotide molecule into a host cell. Such a vector containsheterologous polynucleotide sequences, that is polynucleotide sequencesthat are not naturally found adjacent to polynucleotide molecules of thepresent invention and that preferably are derived from a species otherthan the species from which the polynucleotide molecule(s) are derived.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a transposon (such as described in U.S. Pat. No.5,792,294), a virus or a plasmid.

One type of recombinant vector comprises a polynucleotide molecule ofthe present invention operatively linked to an expression vector. Thephrase “operably linked” refers to insertion of a polynucleotidemolecule into an expression vector in a manner such that the molecule isable to be expressed when transformed into a host cell. As used herein,an expression vector is a DNA or RNA vector that is capable oftransforming a host cell and of effecting expression of a specifiedpolynucleotide molecule. Preferably, the expression vector is alsocapable of replicating within the host cell. Expression vectors can beeither prokaryotic or eukaryotic, and are typically viruses or plasmids.Expression vectors of the present invention include any vectors thatfunction (i.e., direct gene expression) in recombinant cells of thepresent invention, including in bacterial, fungal, endoparasite,arthropod, animal, and plant cells. Particularly preferred expressionvectors of the present invention can direct gene expression in yeastand/or plants cells.

In particular, expression vectors of the present invention containregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of polynucleotide molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude transcription control sequences. Transcription control sequencesare sequences which control the initiation, elongation, and terminationof transcription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art. Particularly preferred transcriptioncontrol sequences are promoters active in directing transcription inplants, either constitutively or stage and/or tissue specific, dependingon the use of the plant or parts thereof. These plant promoters include,but are not limited to, promoters showing constitutive expression, suchas the 35S promoter of Cauliflower Mosaic Virus (CaMV), those forfruit-specific expression, such as the polygalacturonase (PG) promoterfrom tomato.

Recombinant molecules of the present invention may also (a) containsecretory signals (i.e., signal segment nucleic acid sequences) toenable an expressed polypeptide of the present invention to be secretedfrom the cell that produces the polypeptide and/or (b) contain fusionsequences which lead to the expression of nucleic acid molecules of thepresent invention as fusion proteins. Examples of suitable signalsegments include any signal segment capable of directing the secretionof a polypeptide of the present invention. Preferred signal segmentsinclude, but are not limited to, Nicotiana nectarin signal peptide (U.S.Pat. No. 5,939,288), tobacco extensin signal, the soy oleosin oil bodybinding protein signal. In addition, a nucleic acid molecule of thepresent invention can be joined to a fusion segment that directs theencoded polypeptide to the proteosome, such as a ubiquitin fusionsegment. Recombinant molecules may also include intervening and/oruntranslated sequences surrounding and/or within the nucleic acidsequences of nucleic acid molecules of the present invention.

Host Cells

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention. Transformation of a polynucleotidemolecule into a cell can be accomplished by any method by which apolynucleotide molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. Transformed polynucleotidemolecules of the present invention can remain extrachromosomal or canintegrate into one or more sites within a chromosome of the transformed(i.e., recombinant) cell in such a manner that their ability to beexpressed is retained.

Suitable host cells to transform include any cell that can betransformed with a polynucleotide of the present invention. Host cellsof the present invention either can be endogenously (i.e., naturally)capable of producing polypeptides of the present invention or can becapable of producing such polypeptides after being transformed with atleast one polynucleotide molecule of the present invention. Host cellsof the present invention can be any cell capable of producing at leastone protein of the present invention, and include plant, bacterial,fungal (including yeast), parasite, and arthropod cells. Preferably, thehost cell is a plant, arthropod or yeast cell. Non limiting examples ofarthropod cells include insect cells such as Spodoptera frugiperda (Sf)cells, e.g. Sf9, Sf21, Trichoplusia ni cells, and Drosophila S2 cells.

An example of a bacterial cell useful as a host cell of the presentinvention is Synechococcus spp. (also known as Synechocystis spp.), forexample Synechococcus elongatus.

The cells may be of an organism suitable for a fermentation process. Asused herein, the term the “fermentation process” refers to anyfermentation process or any process comprising a fermentation step. Afermentation process includes, without limitation, fermentationprocesses used to produce alcohols (e.g., ethanol, methanol, butanol);organic acids (e.g., citric acid, acetic acid, itaconic acid, lacticacid, gluconic acid); ketones (e.g., acetone); amino acids (e.g.,glutamic acid); gases (e.g., H₂ and CO₂); antibiotics (e.g., penicillinand tetracycline); enzymes; vitamins (e.g., riboflavin, beta-carotene);and hormones. Fermentation processes also include fermentation processesused in the consumable alcohol industry (e.g., beer and wine), dairyindustry (e.g., fermented dairy products), leather industry and tobaccoindustry. Preferred fermentation processes include alcohol fermentationprocesses, as are well known in the art. Preferred fermentationprocesses are anaerobic fermentation processes, as are well known in theart.

Suitable fermenting cells, typically microorganisms are able to ferment,i.e., convert, sugars, such as glucose or maltose, directly orindirectly into the desired fermentation product. Examples of fermentingmicroorganisms include fungal organisms, such as yeast. As used herein,“yeast” includes Saccharomyces spp., Saccharomyces cerevisiae,Saccharomyces carlbergensis, Candida spp., Kluveromyces spp., Pichiaspp., Hansenula spp., Trichoderma spp., Lipomyces starkey, and Yarrowialipolytica. Preferred yeast include strains of the Saccharomyces spp.,and in particular, Saccharomyces cerevisiae. Commercially availableyeast include, e.g., Red Star/Lesaffre Ethanol Red (available from RedStar/Lesaffre, USA) FALI (available from Fleischmann's Yeast, a divisionof Burns Philp Food Inc., USA), SUPERSTART (available from Alltech),GERT STRAND (available from Gert Strand AB, Sweden) and FERMIOL(available from DSM Specialties).

Recombinant DNA technologies can be used to improve expression of atransformed polynucleotide molecule by manipulating, for example, thenumber of copies of the polynucleotide molecule within a host cell, theefficiency with which those polynucleotide molecules are transcribed,the efficiency with which the resultant transcripts are translated, andthe efficiency of post-translational modifications. Recombinanttechniques useful for increasing the expression of polynucleotidemolecules of the present invention include, but are not limited to,operatively linking polynucleotide molecules to high-copy numberplasmids, integration of the polynucleotide molecule into one or morehost cell chromosomes, addition of vector stability sequences toplasmids, substitutions or modifications of transcription controlsignals (e.g., promoters, operators, enhancers), substitutions ormodifications of translational control signals (e.g., ribosome bindingsites, Shine-Dalgarno sequences), modification of polynucleotidemolecules of the present invention to correspond to the codon usage ofthe host cell, and the deletion of sequences that destabilizetranscripts.

Transgenic Plants

The term “plant” as used herein as a noun refers to whole plants, but asused as an adjective refers to any substance which is present in,obtained from, derived from, or related to a plant, such as for example,plant organs (e.g. leaves, stems, roots, flowers), single cells (e.g.pollen), seeds, plant cells and the like. Plants provided by orcontemplated for use in the practice of the present invention includeboth monocotyledons and dicotyledons. In preferred embodiments, theplants of the present invention are crop plants (for example, cerealsand pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet,cassava, barley, or pea), or other legumes. The plants may be grown forproduction of edible roots, tubers, leaves, stems, flowers or fruit. Theplants may be vegetables or ornamental plants. The plants of theinvention may be: corn (Zea mays), canola (Brassica napus, Brassica rapassp.), flax (Linum usitatissimum), alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolour, Sorghumvulgare), sunflower (Helianthus annus), wheat (Tritium aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Lopmoea batatus), cassaya (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus),citris tree (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasenensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifer indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia intergrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), oats, or barley.

In a preferred embodiment, the plant is an angiosperm.

In one embodiment, the plant is an oilseed plant, preferably an oilseedcrop plant. As used herein, an “oilseed plant” is a plant species usedfor the commercial production of oils from the seeds of the plant. Theoilseed plant may be oil-seed rape (such as canola), maize, sunflower,soybean, sorghum, flax (linseed) or sugar beet. Furthermore, the oilseedplant may be other Brassicas, cotton, peanut, poppy, mustard, castorbean, sesame, safflower, or nut producing plants. The plant may producehigh levels of oil in its fruit, such as olive, oil palm or coconut.Horticultural plants to which the present invention may be applied arelettuce, endive, or vegetable brassicas including cabbage, broccoli, orcauliflower. The present invention may be applied in tobacco, cucurbits,carrot, strawberry, tomato, or pepper.

In a further preferred embodiment, the non-transgenic plant used toproduce a transgenic plant of the invention produces oil, especially inthe seed, which has less than 20%, less than 10% or less than 5% 18:2fatty acids and/or which has less than 10% or less than 5% 18:3 fattyacids.

Transgenic plants, as defined in the context of the present inventioninclude plants (as well as parts and cells of said plants) and theirprogeny which have been genetically modified using recombinanttechniques to cause production of at least one polypeptide of thepresent invention in the desired plant or plant organ. Transgenic plantscan be produced using techniques known in the art, such as thosegenerally described in A. Slater et al., Plant Biotechnology—The GeneticManipulation of Plants, Oxford University Press (2003), and P. Christouand H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons(2004).

In a preferred embodiment, the transgenic plants are homozygous for eachand every gene that has been introduced (transgene) so that theirprogeny do not segregate for the desired phenotype. The transgenicplants may also be heterozygous for the introduced transgene(s), suchas, for example, in F1 progeny which have been grown from hybrid seed.Such plants may provide advantages such as hybrid vigour, well known inthe art.

The transgenic plants may also comprise additional transgenes involvedin the production of LC-PUFAs such as, but not limited to, a Δ6desaturase, a Δ9 elongase, a desaturase, a Δ6 elongase, a Δ5 desaturasewith activity on a 20:3 substrate, an omega-desaturase, a Δ9 elongase, aΔ4 desaturase, a Δ7 elongase and/or members of the polyketide synthasepathway. Examples of such enzymes are known in the art and include thosedescribed in WO 05/103253 (see, for example, Table 1 of WO 05/103253).

A polynucleotide of the present invention may be expressedconstitutively in the transgenic plants during all stages ofdevelopment. Depending on the use of the plant or plant organs, thepolypeptides may be expressed in a stage-specific manner. Furthermore,the polynucleotides may be expressed tissue-specifically.

Regulatory sequences which are known or are found to cause expression ofa gene encoding a polypeptide of interest in plants may be used in thepresent invention. The choice of the regulatory sequences used dependson the target plant and/or target organ of interest. Such regulatorysequences may be obtained from plants or plant viruses, or may bechemically synthesized. Such regulatory sequences are well known tothose skilled in the art.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

A number of constitutive promoters that are active in plant cells havebeen described. Suitable promoters for constitutive expression in plantsinclude, but are not limited to, the cauliflower mosaic virus (CaMV) 35Spromoter, the Figwort mosaic virus (FMV) 35S, the sugarcane bacilliformvirus promoter, the commelina yellow mottle virus promoter, thelight-inducible promoter from the small subunit of theribulose-1,5-bis-phosphate carboxylase, the rice cytosolictriosephosphate isomerase promoter, the adeninephosphoribosyltransferase promoter of Arabidopsis, the rice actin 1 genepromoter, the mannopine synthase and octopine synthase promoters, theAdh promoter, the sucrose synthase promoter, the R gene complexpromoter, and the chlorophyll α/β binding protein gene promoter. Thesepromoters have been used to create DNA vectors that have been expressedin plants; see, e.g., PCT publication WO 8402913. All of these promotershave been used to create various types of plant-expressible recombinantDNA vectors.

For the purpose of expression in source tissues of the plant, such asthe leaf, seed, root or stem, it is preferred that the promotersutilized in the present invention have relatively high expression inthese specific tissues. For this purpose, one may choose from a numberof promoters for genes with tissue- or cell-specific or -enhancedexpression. Examples of such promoters reported in the literatureinclude the chloroplast glutamine synthetase GS2 promoter from pea, thechloroplast fructose-1,6-biphosphatase promoter from wheat, the nuclearphotosynthetic ST-LS1 promoter from potato, the serine/threonine kinasepromoter and the glucoamylase (CHS) promoter from Arabidopsis thaliana.Also reported to be active in photosynthetically active tissues are theribulose-1,5-bisphosphate carboxylase promoter from eastern larch (Larixlaricina), the promoter for the Cab gene, Cab6, from pine, the promoterfor the Cab-1 gene from wheat, the promoter for the Cab-1 gene fromspinach, the promoter for the Cab 1R gene from rice, the pyruvate,orthophosphate dikinase (PPDK) promoter from Zea mays, the promoter forthe tobacco Lhcb1*2 gene, the Arabidopsis thaliana Suc2 sucrose-H³⁰symporter promoter, and the promoter for the thylakoid membrane proteingenes from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS).

Other promoters for the chlorophyll α/β-binding proteins may also beutilized in the present invention, such as the promoters for LhcB geneand PsbP gene from white mustard (Sinapis alba). A variety of plant genepromoters that are regulated in response to environmental, hormonal,chemical, and/or developmental signals, also can be used for expressionof RNA-binding protein genes in plant cells, including promotersregulated by (1) heat, (2) light (e.g., pea RbcS-3A promoter, maize RbcSpromoter); (3) hormones, such as abscisic acid, (4) wounding (e.g.,WunI); or (5) chemicals, such as methyl jasminate, salicylic acid,steroid hormones, alcohol, Safeners (WO 9706269), or it may also beadvantageous to employ (6) organ-specific promoters.

For the purpose of expression in sink tissues of the plant, such as thetuber of the potato plant, the fruit of tomato, or the seed of soybean,canola, cotton, Zea mays, wheat, rice, and barley, it is preferred thatthe promoters utilized in the present invention have relatively highexpression in these specific tissues. A number of promoters for geneswith tuber-specific or -enhanced expression are known, including theclass I patatin promoter, the promoter for the potato tuber ADPGPPgenes, both the large and small subunits, the sucrose synthase promoter,the promoter for the major tuber proteins including the 22 kD proteincomplexes and proteinase inhibitors, the promoter for the granule boundstarch synthase gene (GBSS), and other class I and II patatinspromoters. Other promoters can also be used to express a protein inspecific tissues, such as seeds or fruits. The promoter forβ-conglycinin or other seed-specific promoters such as the napin andphaseolin promoters, can be used. A particularly preferred promoter forZea mays endosperm expression is the promoter for the glutelin gene fromrice, more particularly the Osgt-1 promoter. Examples of promoterssuitable for expression in wheat include those promoters for theADPglucose pyrosynthase (ADPGPP) subunits, the granule bound and otherstarch synthase, the branching and debranching enzymes, theembryogenesis-abundant proteins, the gliadins, and the glutenins.Examples of such promoters in rice include those promoters for theADPGPP subunits, the granule bound and other starch synthase, thebranching enzymes, the debranching enzymes, sucrose synthases, and theglutelins. A particularly preferred promoter is the promoter for riceglutelin, Osgt-1 gene. Examples of such promoters for barley includethose for the ADPGPP subunits, the granule bound and other starchsynthase, the branching enzymes, the debranching enzymes, sucrosesynthases, the hordeins, the embryo globulins, and the aleurone specificproteins.

Root specific promoters may also be used. An example of such a promoteris the promoter for the acid chitinase gene. Expression in root tissuecould also be accomplished by utilizing the root specific subdomains ofthe CaMV 35S promoter that have been identified.

In a particularly preferred embodiment, the promoter directs expressionin tissues and organs in which fatty acid and oil biosynthesis takeplace, particularly in seed cells such as endosperm cells and cells ofthe developing embryo. Promoters which are suitable are the oilseed rapenapin gene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USPpromoter (Baumlein et al., 1991), the Arabidopsis oleosin promoter (WO98/45461), the Phaseolus vulgaris phaseolin promoter (U.S. Pat. No.5,504,200), the Brassica Bce4 promoter (WO 91/13980) or the legumin B4promoter (Baumlein et al., 1992), and promoters which lead to theseed-specific expression in monocots such as maize, barley, wheat, rye,rice and the like. Notable promoters which are suitable are the barleylpt2 or lpt1 gene promoter (WO 95/15389 and WO 95/23230) or thepromoters described in WO 99/16890 (promoters from the barley hordeingene, the rice glutelin gene, the rice oryzin gene, the rice prolamingene, the wheat gliadin gene, the wheat glutelin gene, the maize zeingene, the oat glutelin gene, the sorghum kasirin gene, the rye secalingene). Other promoters include those described by Broun et al. (1998)and US 20030159173.

The 5′ non-translated leader sequence can be derived from the promoterselected to express the heterologous gene sequence of the polynucleotideof the present invention, and can be specifically modified if desired soas to increase translation of mRNA. For a review of optimizingexpression of transgenes, see Koziel et al. (1996). The 5′non-translated regions can also be obtained from plant viral RNAs(Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus,Alfalfa mosaic virus, among others) from suitable eukaryotic genes,plant genes (wheat and maize chlorophyll a/b binding protein geneleader), or from a synthetic gene sequence. The present invention is notlimited to constructs wherein the non-translated region is derived fromthe 5′ non-translated sequence that accompanies the promoter sequence.The leader sequence could also be derived from an unrelated promoter orcoding sequence. Leader sequences useful in context of the presentinvention comprise the maize Hsp70 leader (U.S. Pat. No. 5,362,865 andU.S. Pat. No. 5,859,347), and the TMV omega element.

The termination of transcription is accomplished by a 3′ non-translatedDNA sequence operably linked in the chimeric vector to thepolynucleotide of interest. The 3′ non-translated region of arecombinant DNA molecule contains a polyadenylation signal thatfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. The 3′ non-translated region can be obtained fromvarious genes that are expressed in plant cells. The nopaline synthase3′ untranslated region, the 3′ untranslated region from pea smallsubunit Rubisco gene, the 3′ untranslated region from soybean 7S seedstorage protein gene are commonly used in this capacity. The 3′transcribed, non-translated regions containing the polyadenylate signalof Agrobacterium tumor-inducing (Ti) plasmid genes are also suitable.

Four general methods for direct delivery of a gene into cells have beendescribed: (1) chemical methods (Graham et al., 1973); (2) physicalmethods such as microinjection (Capecchi, 1980); electroporation (see,for example, WO 87/06614, U.S. Pat. Nos. 5,472,869, 5,384,253, WO92/09696 and WO 93/21335); and the gene gun (see, for example, U.S. Pat.No. 4,945,050 and U.S. Pat. No. 5,141,131); (3) viral vectors (Clapp,1993; Lu et al., 1993; Eglitis et al., 1988); and (4) receptor-mediatedmechanisms (Curiel et al., 1992; Wagner et al., 1992).

Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. A particular advantage ofmicroprojectile bombardment, in addition to it being an effective meansof reproducibly transforming monocots, is that neither the isolation ofprotoplasts, nor the susceptibility of Agrobacterium infection arerequired. An illustrative embodiment of a method for delivering DNA intoZea mays cells by acceleration is a biolistics α-particle deliverysystem, that can be used to propel particles coated with DNA through ascreen, such as a stainless steel or Nytex screen, onto a filter surfacecovered with corn cells cultured in suspension. A particle deliverysystem suitable for use with the present invention is the heliumacceleration PDS-1000/He gun available from Bio-Rad Laboratories.

For the bombardment, cells in suspension may be concentrated on filters.Filters containing the cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between the gun and thecells to be bombarded.

Alternatively, immature embryos or other target cells may be arranged onsolid culture medium. The cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between theacceleration device and the cells to be bombarded. Through the use oftechniques set forth herein one may obtain up to 1000 or more foci ofcells transiently expressing a marker gene. The number of cells in afocus that express the exogenous gene product 48 hours post-bombardmentoften range from one to ten and average one to three.

In bombardment transformation, one may optimize the pre-bombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

In another alternative embodiment, plastids can be stably transformed.Methods disclosed for plastid transformation in higher plants includeparticle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (U.S. Pat. No. 5,451,513, U.S. Pat. No. 5,545,818, U.S.Pat. No. 5,877,402, U.S. Pat. No. 5,932,479, and WO 99/05265).

Accordingly, it is contemplated that one may wish to adjust variousaspects of the bombardment parameters in small scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors bymodifying conditions that influence the physiological state of therecipient cells and that may therefore influence transformation andintegration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. The execution of otherroutine adjustments will be known to those of skill in the art in lightof the present disclosure.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (see, for example, U.S. Pat. No. 5,177,010, U.S. Pat.No. 5,104,310, U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135).Further, the integration of the T-DNA is a relatively precise processresulting in few rearrangements. The region of DNA to be transferred isdefined by the border sequences, and intervening DNA is usually insertedinto the plant genome.

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., In: Plant DNA InfectiousAgents, Hohn and Schell, eds., Springer-Verlag, New York, pp. 179-203(1985). Moreover, technological advances in vectors forAgrobacterium-mediated gene transfer have improved the arrangement ofgenes and restriction sites in the vectors to facilitate construction ofvectors capable of expressing various polypeptide coding genes. Thevectors described have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Inaddition, Agrobacterium containing both armed and disarmed Ti genes canbe used for the transformations. In those plant varieties whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single genetic locus on one chromosome. Suchtransgenic plants can be referred to as being hemizygous for the addedgene. More preferred is a transgenic plant that is homozygous for theadded gene; i.e., a transgenic plant that contains two added genes, onegene at the same locus on each chromosome of a chromosome pair. Ahomozygous transgenic plant can be obtained by sexually mating (selfing)an independent segregant transgenic plant that contains a single addedgene, germinating some of the seed produced and analyzing the resultingplants for the gene of interest.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both exogenous genes. Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in Fehr, In: Breeding Methods for Cultivar Development,Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. Application ofthese systems to different plant varieties depends upon the ability toregenerate that particular plant strain from protoplasts. Illustrativemethods for the regeneration of cereals from protoplasts are described(Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).

Other methods of cell transformation can also be used and include butare not limited to introduction of DNA into plants by direct DNAtransfer into pollen, by direct injection of DNA into reproductiveorgans of a plant, or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., In: Methods for Plant MolecularBiology, Academic Press, San Diego, Calif., (1988). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredexogenous nucleic acid is cultivated using methods well known to oneskilled in the art.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135, U.S. Pat. No.5,518,908); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011);Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al., 1996); and pea(Grant et al., 1995).

Methods for transformation of cereal plants such as wheat and barley forintroducing genetic variation into the plant by introduction of anexogenous nucleic acid and for regeneration of plants from protoplastsor immature plant embryos are well known in the art, see for example,Canadian Patent Application No. 2,092,588, Australian Patent ApplicationNo 61781/94, Australian Patent No 667939, U.S. Pat. No. 6,100,447,International Patent Application PCT/US97/10621, U.S. Pat. No.5,589,617, U.S. Pat. No. 6,541,257, and other methods are set out inpatent specification WO99/14314. Preferably, transgenic wheat or barleyplants are produced by Agrobacterium tumefaciens mediated transformationprocedures. Vectors carrying the desired nucleic acid construct may beintroduced into regenerable wheat cells of tissue cultured plants orexplants, or suitable plant systems such as protoplasts.

The regenerable wheat cells are preferably from the scutellum ofimmature embryos, mature embryos, callus derived from these, or themeristematic tissue.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeWestern blot and enzyme assay. One particularly useful way to quantitateprotein expression and to detect replication in different plant tissuesis to use a reporter gene, such as GUS. Once transgenic plants have beenobtained, they may be grown to produce plant tissues or parts having thedesired phenotype. The plant tissue or plant parts, may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants with tissues or parts having the desiredcharacteristics.

Transgenic Hon-Human Animals

A “transgenic non-human animal” refers to an animal, other than a human,that contains a gene construct (“transgene”) not found in a wild-typeanimal of the same species or breed. A “transgene” as referred to hereinhas the normal meaning in the art of biotechnology and includes agenetic sequence which has been produced or altered by recombinant DNAor RNA technology and which has been introduced into an animal cell. Thetransgene may include genetic sequences derived from an animal cell.Typically, the transgene has been introduced into the animal by humanmanipulation such as, for example, by transformation but any method canbe used as one of skill in the art recognizes.

Techniques for producing transgenic animals are well known in the art. Auseful general textbook on this subject is Houdebine, Transgenicanimals—Generation and Use (Harwood Academic, 1997).

Heterologous DNA can be introduced, for example, into fertilizedmammalian ova. For instance, totipotent or pluripotent stem cells can betransformed by microinjection, calcium phosphate mediated precipitation,liposome fusion, retroviral infection or other means, the transformedcells are then introduced into the embryo, and the embryo then developsinto a transgenic animal. In a highly preferred method, developingembryos are infected with a retrovirus containing the desired DNA, andtransgenic animals produced from the infected embryo. In a mostpreferred method, however, the appropriate DNAs are coinjected into thepronucleus or cytoplasm of embryos, preferably at the single cell stage,and the embryos allowed to develop into mature transgenic animals.

Another method used to produce a transgenic animal involvesmicroinjecting a nucleic acid into pro-nuclear stage eggs by standardmethods. Injected eggs are then cultured before transfer into theoviducts of pseudopregnant recipients.

Transgenic animals may also be produced by nuclear transfer technology.Using this method, fibroblasts from donor animals are stably transfectedwith a plasmid incorporating the coding sequences for a binding domainor binding partner of interest under the control of regulatorysequences. Stable transfectants are then fused to enucleated oocytes,cultured and transferred into female recipients.

Feedstuffs

The present invention includes compositions which can be used asfeedstuffs. For purposes of the present invention, “feedstuffs” includeany food or preparation for human or animal consumption (including forenteral and/or parenteral consumption) which when taken into the body(a) serve to nourish or build up tissues or supply energy; and/or (b)maintain, restore or support adequate nutritional status or metabolicfunction. Feedstuffs of the invention include nutritional compositionsfor babies and/or young children.

Feedstuffs of the invention comprise, for example, a cell of theinvention, a plant of the invention, the plant part of the invention,the seed of the invention, an extract of the invention, the product ofthe method of the invention, the product of the fermentation process ofthe invention, or a composition along with a suitable carrier(s). Theterm “carrier” is used in its broadest sense to encompass any componentwhich may or may not have nutritional value. As the skilled addresseewill appreciate, the carrier must be suitable for use (or used in asufficiently low concentration) in a feedstuff such that it does nothave deleterious effect on an organism which consumes the feedstuff.

The feedstuff of the present invention comprises an oil, fatty acidester, or fatty acid produced directly or indirectly by use of themethods, cells or plants disclosed herein. The composition may either bein a solid or liquid form. Additionally, the composition may includeedible macronutrients, vitamins, and/or minerals in amounts desired fora particular use. The amounts of these ingredients will vary dependingon whether the composition is intended for use with normal individualsor for use with individuals having specialized needs, such asindividuals suffering from metabolic disorders and the like.

Examples of suitable carriers with nutritional value include, but arenot limited to, macronutrients such as edible fats, carbohydrates andproteins. Examples of such edible fats include, but are not limited to,coconut oil, borage oil, fungal oil, black current oil, soy oil, andmono- and diglycerides. Examples of such carbohydrates include (but arenot limited to): glucose, edible lactose, and hydrolyzed search.Additionally, examples of proteins which may be utilized in thenutritional composition of the invention include (but are not limitedto) soy proteins, electrodialysed whey, electrodialysed skim milk, milkwhey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to thefeedstuff compositions of the present invention: calcium, phosphorus,potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc,selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other suchvitamins and minerals may also be added.

The components utilized in the feedstuff compositions of the presentinvention can be of semi-purified or purified origin. By semi-purifiedor purified is meant a material which has been prepared by purificationof a natural material or by de novo synthesis.

A feedstuff composition of the present invention may also be added tofood even when supplementation of the diet is not required. For example,the composition may be added to food of any type, including (but notlimited to): margarine, modified butter, cheeses, milk, yogurt,chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats,fish and beverages.

The genus Saccharomyces spp is used in both brewing of beer and winemaking and also as an agent in baking, particularly bread. Yeast is amajor constituent of vegetable extracts. Yeast is also used as anadditive in animal feed. It will be apparent that genetically engineeredyeast strains can be provided which are adapted to synthesise LC-PUFA asdescribed herein. These yeast strains can then be used in food stuffsand in wine and beer making to provide products which have enhancedfatty acid content.

Additionally, fatty acids produced in accordance with the presentinvention or host cells transformed to contain and express the subjectgenes may also be used as animal food supplements to alter an animal'stissue or milk fatty acid composition to one more desirable for human oranimal consumption. Examples of such animals include sheep, cattle,horses and the like.

Furthermore, feedstuffs of the invention can be used in aquaculture toincrease the levels of fatty acids in fish for human or animalconsumption.

Compositions

The present invention also encompasses compositions, particularlypharmaceutical compositions, comprising one or more of the fatty acidsand/or resulting oils produced using the methods of the invention.

A pharmaceutical composition may comprise one or more of the fatty acidsand/or oils, in combination with a standard, well-known, non-toxicpharmaceutically-acceptable carrier, adjuvant or vehicle such asphosphate-buffered saline, water, ethanol, polyols, vegetable oils, awetting agent or an emulsion such as a water/oil emulsion. Thecomposition may be in either a liquid or solid form. For example, thecomposition may be in the form of a tablet, capsule, ingestible liquidor powder, injectible, or topical ointment or cream. Proper fluidity canbe maintained, for example, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants. It mayalso be desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Besides such inert diluents, thecomposition can also include adjuvants, such as wetting agents,emulsifying and suspending agents, sweetening agents, flavoring agentsand perfuming agents.

Suspensions, in addition to the active compounds, may comprisesuspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanthor mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared usingtechniques well known in the art. For example, fatty acids produced inaccordance with the present invention can be tableted with conventionaltablet bases such as lactose, sucrose, and cornstarch in combinationwith binders such as acacia, cornstarch or gelatin, disintegratingagents such as potato starch or alginic acid, and a lubricant such asstearic acid or magnesium stearate. Capsules can be prepared byincorporating these excipients into a gelatin capsule along withantioxidants and the relevant fatty acid(s).

For intravenous administration, the fatty acids produced in accordancewith the present invention or derivatives thereof may be incorporatedinto commercial formulations.

A typical dosage of a particular fatty acid is from 0.1 mg to 20 g,taken from one to five times per day (up to 100 g daily) and ispreferably in the range of from about 10 mg to about 1, 2, 5, or 10 gdaily (taken in one or multiple doses). As known in the art, a minimumof about 300 mg/day of fatty acid, especially LC-PUFA, is desirable.However, it will be appreciated that any amount of fatty acid will bebeneficial to the subject.

Possible routes of administration of the pharmaceutical compositions ofthe present invention include, for example, enteral (e.g., oral andrectal) and parenteral. For example, a liquid preparation may beadministered orally or rectally. Additionally, a homogenous mixture canbe completely dispersed in water, admixed under sterile conditions withphysiologically acceptable diluents, preservatives, buffers orpropellants to form a spray or inhalant.

The dosage of the composition to be administered to the patient may bedetermined by one of ordinary skill in the art and depends upon variousfactors such as weight of the patient, age of the patient, overallhealth of the patient, past history of the patient, immune status of thepatient, etc.

Additionally, the compositions of the present invention may be utilizedfor cosmetic purposes. It may be added to pre-existing cosmeticcompositions such that a mixture is formed or a fatty acid producedaccording to the subject invention may be used as the sole “active”ingredient in a cosmetic composition.

Production of Oils

Techniques that are routinely practiced in the art can be used toextract, process, and analyze the oils produced by cells, plants, seeds,etc of the instant invention. Typically, plant seeds are cooked,pressed, and extracted to produce crude oil, which is then degummed,refined, bleached, and deodorized. Generally, techniques for crushingseed are known in the art. For example, oilseeds can be tempered byspraying them with water to raise the moisture content to, e.g., 8.5%,and flaked using a smooth roller with a gap setting of 0.23 to 0.27 mm.Depending on the type of seed, water may not be added prior to crushing.Application of heat deactivates enzymes, facilitates further cellrupturing, coalesces the oil droplets, and agglomerates proteinparticles, all of which facilitate the extraction process.

The majority of the seed oil is released by passage through a screwpress. Cakes expelled from the screw press are then solvent extracted,e.g., with hexane, using a heat traced column. Alternatively, crude oilproduced by the pressing operation can be passed through a settling tankwith a slotted wire drainage top to remove the solids that are expressedwith the oil during the pressing operation. The clarified oil can bepassed through a plate and frame filter to remove any remaining finesolid particles. If desired, the oil recovered from the extractionprocess can be combined with the clarified oil to produce a blendedcrude oil.

Once the solvent is stripped from the crude oil, the pressed andextracted portions are combined and subjected to normal oil processingprocedures (i.e., degumming, caustic refining, bleaching, anddeodorization). Degumming can be performed by addition of concentratedphosphoric acid to the crude oil to convert non-hydratable phosphatidesto a hydratable form, and to chelate minor metals that are present. Gumis separated from the oil by centrifugation. The oil can be refined byaddition of a sufficient amount of a sodium hydroxide solution totitrate all of the fatty acids and removing the soaps thus formed.

Deodorization can be performed by heating the oil to 260° C. undervacuum, and slowly introducing steam into the oil at a rate of about 0.1ml/minute/100 ml of oil. After about 30 minutes of sparging, the oil isallowed to cool under vacuum. The oil is typically transferred to aglass container and flushed with argon before being stored underrefrigeration. If the amount of oil is limited, the oil can be placedunder vacuum, e.g., in a Parr reactor and heated to 260° C. for the samelength of time that it would have been deodorized. This treatmentimproves the color of the oil and removes a majority of the volatilesubstances.

Antibodies

The invention also provides monoclonal or polyclonal antibodies topolypeptides of the invention or fragments thereof. Thus, the presentinvention further provides a process for the production of monoclonal orpolyclonal antibodies to polypeptides of the invention.

The term “binds specifically” refers to the ability of the antibody tobind to proteins of the present invention but not other knowndesaturase, conjugase, epoxidase and/or hydroxylase-like polypeptides.

As used herein, the term “epitope” refers to a region of a polypeptideof the invention which is bound by the antibody. An epitope can beadministered to an animal to generate antibodies against the epitope,however, antibodies of the present invention preferably specificallybind the epitope region in the context of the entire polypeptide.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptidesuch as those provided as SEQ ID NOs: 16 to 30, 73 to 78, 80 and 134.Serum from the immunised animal is collected and treated according toknown procedures. If serum containing polyclonal antibodies containsantibodies to other antigens, the polyclonal antibodies can be purifiedby immunoaffinity chromatography. Techniques for producing andprocessing polyclonal antisera are known in the art. In order that suchantibodies may be made, the invention also provides peptides of theinvention or fragments thereof haptenised to another peptide for use asimmunogens in animals.

Monoclonal antibodies directed against polypeptides of the invention canalso be readily produced by one skilled in the art. The generalmethodology for making monoclonal antibodies by hybridomas is wellknown. Immortal antibody-producing cell lines can be created by cellfusion, and also by other techniques such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.Panels of monoclonal antibodies produced can be screened for variousproperties; i.e., for isotype and epitope affinity.

An alternative technique involves screening phage display librarieswhere, for example the phage express scFv fragments on the surface oftheir coat with a large variety of complementarity determining regions(CDRs). This technique is well known in the art.

For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes fragments of whole antibodies whichretain their binding activity for a target antigen. Such fragmentsinclude Fv, F(ab′) and F(ab′)₂ fragments, as well as single chainantibodies (scFv). Furthermore, the antibodies and fragments thereof maybe humanised antibodies, for example as described in EP-A-239400.

Antibodies of the invention may be bound to a solid support and/orpackaged into kits in a suitable container along with suitable reagents,controls, instructions and the like.

In an embodiment, antibodies of the present invention are detectablylabeled. Exemplary detectable labels that allow for direct measurementof antibody binding include radiolabels, fluorophores, dyes, magneticbeads, chemiluminescers, colloidal particles, and the like. Examples oflabels which permit indirect measurement of binding include enzymeswhere the substrate may provide for a coloured or fluorescent product.Additional exemplary detectable labels include covalently bound enzymescapable of providing a detectable product signal after addition ofsuitable substrate. Examples of suitable enzymes for use in conjugatesinclude horseradish peroxidase, alkaline phosphatase, malatedehydrogenase and the like. Where not commercially available, suchantibody-enzyme conjugates are readily produced by techniques known tothose skilled in the art. Further exemplary detectable labels includebiotin, which binds with high affinity to avidin or streptavidin;fluorochromes (e.g., phycobiliproteins, phypoerythrin andallophycocyanins; fluorescein and Texas red), which can be used with afluorescence activated cell sorter; haptens; and the like. Preferably,the detectable label allows for direct measurement in a plateluminometer, e.g., biotin. Such labeled antibodies can be used intechniques known in the art to detect polypeptides of the invention.

EXAMPLES Example 1 Identification of Desaturases in the Triboliumcastaneum Genome

Sixty-four published full length insect desaturase protein sequencesavailable at the end of 2005 were aligned using the Clustal X program(Thompson et al., 1997). From this sequence alignment, a highlyconserved peptide sequence was selected (HNYHHAYPWDYKAAEIGMPLNSTASLIRLCASLGWAYDLKSV (SEQ ID NO: 31)) and used to search the Triboliumcastaneum genome using TBLASTN program (Altschul et al, 1997). From thisanalysis, 15 desaturase-like sequences were identified which have beentermed, Tribdesat 1 (coding sequence provided as SEQ ID NO:1, amino acidsequence provided as SEQ ID NO:16), Tribdesat2a (coding sequenceprovided as SEQ ID NO:2, amino acid sequence provided as SEQ ID NO:17),Tribdesat2b (coding sequence provided as SEQ ID NO:3, amino acidsequence provided as SEQ ID NO:18), Tribdesat2c (coding sequenceprovided as SEQ ID NO:4, amino acid sequence provided as SEQ ID NO:19),Tribdesat3 (coding sequence provided as SEQ ID NO:5, amino acid sequenceprovided as SEQ ID NO:20), Tribdesat4 (coding sequence provided as SEQID NO:6, amino acid sequence provided as SEQ ID NO:21), Tribdesat5(coding sequence provided as SEQ ID NO:7, amino acid sequence providedas SEQ ID NO:22), Tribdesat6a (coding sequence provided as SEQ ID NO:8,amino acid sequence provided as SEQ ID NO:23), Tribdesat6b (codingsequence provided as SEQ ID NO:9, amino acid sequence provided as SEQ IDNO:24), Tribdesat7a (coding sequence provided as SEQ ID NO:10, aminoacid sequence provided as SEQ ID NO:25), Tribdesat7b (coding sequenceprovided as SEQ ID NO:11, amino acid sequence provided as SEQ ID NO:26),Tribdesat8 (coding sequence provided as SEQ ID NO:12, amino acidsequence provided as SEQ ID NO:27), Tribdesat10 (coding sequenceprovided as SEQ ID NO:13, amino acid sequence provided as SEQ ID NO:28),Tribdesat11 (coding sequence provided as SEQ ID NO:14, amino acidsequence provided as SEQ ID NO:29) and Tribdesat12 (coding sequenceprovided as SEQ ID NO:15, amino acid sequence provided as SEQ ID NO:30).The sequences listed above as Tribdesat2a, 2b, 2c, 3, 4, 5, 6a, 8, 10,11, 12 were corrected on the basis of cloning and sequencing of thecorresponding cDNAs (see Examples 2-5).

Each of the contigs was then subjected to a gene prediction program inSoftberry (http://www.softberry.com/cgi-bin/programs/gfin/fgenesh).Parameters for Drosophila, Anopheles, C. elegans and Brugia malaya weretested for their ability to predict desaturase genes. For each of thegene predictions, the proteins were subjected to a BLASTP analysisagainst all proteins in the NCBI database to determine if they resembleddesaturases. Neither Anopheles nor Drosophila parameters gave thecorrect prediction for desaturases except for Tribdesat10 which usedAnopheles parameters. For some of the desaturases, parameters forpredicting genes in C. elegans or B. malaya predicted genes similar todesaturases. For three of the desaturases, none of the predictions usingany of the default parameters predicted desaturase genes (Tribdesat4,Tribdesat7a and Tribdesat7b).

For three of the desaturase genes (2 contigs) that could not bepredicted using the gene prediction programs, the genomic regions weresubjected to a BLASTX comparison with all proteins in the NCBI database.The most 5′ region of the genomic fragment resembling desaturases wasnoted and DNA sequences upstream were translated in silico and visuallyexamined for a methionine residue. This was taken to be the startmethionine of the protein sequence. The 3′ region of the genomicfragment that resembled desaturases were examined in a similar mannerfor a stop codon. Each of these protein sequences were visuallyinspected for all the conserved motifs of desaturases (Table 2). ForTribdesat4, no reasonable prediction of the N-terminus or C-terminus ofthe protein could be made due to incomplete sequence of the genome inthese regions.

Each protein sequence was also examined for the presence of three“histidine boxes” (His boxes) which are motifs that are stronglyconserved in all desaturases. The histidine residues in these boxes arethought to be involved in binding iron atoms required for desaturaseactivity. The amino acid positions and sequences of the His boxes in theproteins and two others from Chauliognathus lugubris (see Example 5) arelisted in Table 3, after correction of the predicted protein sequencesfrom analysis of the cDNA clones (Examples 2-5).

TABLE 2 Presence (✓) or absence (X) of conserved desaturase motifs inidentified Tribolium amino acid sequences. Predicted protein Conservedsequence motif Identified Gene size (amino acids) 1 2 3 4 5 6 7 8Tribdesat1 348 ✓ ✓ ✓ X ✓ ✓ ✓ ✓ Tribdesat2a 320 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓Tribdesat2b 297 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Tribdesat2c 321 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓Tribdesat3 286 ✓ X ✓ ✓ ✓ ✓ ✓ ✓ Tribdesat5 353 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓Tribdesat6a 350 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Tribdesat6b 289 ✓ X X ✓ X X ✓ XTribdesat8 374 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Tribdesat10 366 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓Tribdesat11 323 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Tribdesat12 455 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ (Note:Motifs in the sequences may not be identical to the correspondingconserved sequence motif). Concensus amino acid sequences of motifs: 1,AGAHRLW (SEQ ID NO: 104); 2, SETDAD (SEQ ID NO: 105); 3, FFFSHVG (SEQ IDNO: 106); 4, QKKY (SEQ ID NO: 107); 5, NSAAH (SEQ ID NO: 108); 6,GEGWHNYHH (SEQ ID NO: 109); 7, PWDY (SEQ ID NO: 110); 8, GWAY (SEQ IDNO: 111).

TABLE 3 Amino acid location and sequences of histidine boxes inTribolium and soldier beetle desaturase sequences. The number for eachbox corresponds to the location in the protein of the first amino acidresidue of the motif. First HIS box Second HIS box Third HIS boxTribdesat1 90; HRLWTH 109; HRVHH 249; HNYHH (SEQ ID NO: 112) (SEQ ID NO:116) (SEQ ID NO: 121) Tribdesat2a 60; HRLWSH 97; HRAHH 238; HNYHH (SEQID NO: 113) (SEQ ID NO: 117) Tribdesat2b 62; HRLWAH 99; HRIHH 240; HNYHH(SEQ ID NO: 114) Tribdesat2c 55; HRLWAH 92; HRVHH 233; HNYHH Tribdesat365; HRLWAH 102; HQVHH 243; HNYHH (SEQ ID NO: 118) Tribdesat4 96; HRLWSH133; HRVHH 274; HNYHH Tribdesat5 88; HRLWAH 125; HRVHH 265; HNYHHTribdesat6a 88; HRLWAH 125; HRVHH 265; HNYHH Tribdesat6b 15; HRLWAH 52;HRLHH 189; WISYH (SEQ ID NO: 119) (SEQ ID NO: 122) Tribdesat7a 57;HRLWSH 94; HRAHH 234; HNYHH Tribdesat8 93; HRLWAH 130; HRVHH 271; HNYHHTribdesat10 75; HRLWAH 112; HRVHH 254; HNYHH Tribdesat11 77; HRLYSH 114;HRQHH 255; HNYHH (SEQ ID NO: 115) (SEQ ID NO: 120) Tribdesat12 104;HRLWAH 141; HRVHH 282; HNYHH CL1 (SEQ ID NO: 73) 88; HRLWAH 125; HRVHH265; HNYHH CL3 (SEQ ID NO: 74) 88; HRLWSH 125; HRVHH 265; HNYHH

Example 2 Cloning of Tribdesat4

The inventors attempted to obtain Tribdesat4 using inverse PCR on cDNAas we could not predict from the genome sequence either the beginning orthe end of the gene. First strand synthesis was performed by mixing 3 μllarval RNA (24 μg), 100 pmol of each of the oligonucleotide primers fromClontech, Smart IV Oligo 5′AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3′(SEQ ID NO: 32); and CDS/3′ (5′ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30VN)(N=A, G, C or T; V=A, G or C (SEQ ID NO: 33)). This was incubated at 72°C. for 2 min after which it was immediately cooled on ice. To thecontents of the tube was added 2 μl 5× first strand buffer (Powerscript;Clontech), 1 μl DTT (20 mM), 1 μl dNTP mix (final concentration of 200μM each dNTP) and 1 μl Powerscript reverse transcriptase (Clontech).First strand cDNA synthesis was allowed to occur at 42° C. for 1 hour,after which it was immediately cooled on ice. NaOH (1 μl, 25 mM) wasadded and incubated at 68° C. for 30 min. An aliquot of the first strandsynthesis (6 μl) was mixed with 5 μl 10× Advantage 2 Buffer (Clontech),1 μl 50× dNTP mix, 1 μl CDS/5 oligo (5′AAGCAGTGGTATCAACGCAGAGT; Clontech(SEQ ID NO: 34)), 1 μl CDS/3 oligo, 1 μl 50× Advantage 2 Polymerase andmade to a final volume of 50 μl. The mixture then went through atemperature regime as follows: 72° C./10 min; 95° C./1 min; then 3cycles of 95° C./15 sec, 68° C./8 min.

An aliquot (5 μl) was separated on a 1% agarose gel to ensure thatamplification had occurred. The sample was then digested with SfiI for 2hours at 50° C. and the restriction enzyme removed using the QIAgen PCRpurification kit according to the manufacturer's instructions. Theeluted sample was diluted for ligation to ensure intramolecularligations rather than intermolecular ligation. Digested DNA (0.5 μg) wasdiluted in a ligation mixture of 500 μl and incubated overnight at 16°C. The ligation was concentrated by glycogen precipitation (150 μgglycogen, 1.4 ml ice-cold 95% ethanol at −80° C. for 2 hours) afterwhich DNA was pelleted by centrifugation (12 000 g, 20 min). The DNA wasresuspended in 10 μl sterile water. The inverse PCR reaction wasperformed on 1 μl of this precipitated DNA and included 5 μl 10×Advantage 2 PCR Buffer (Clontech), 10 pmol Desat4 GSP1(5′ATTATGAGCGGATCGGCTTCCAAGTC (SEQ ID NO: 35), 10 pmol Desat4 GSP2(5′CGAAACAATTTGGAATTCGTTTTGGG (SEQ ID NO: 36), 200 μM each dNTP, 1 μl50× BD Advantage 2 Polymerase and sterile water to a final volume of 50μl. The PCR conditions were as follows: 95° C./1 min, then 30 cycles of95° C./30 sec, 68° C./3 min, then 68° C./3 min.

An aliquot (5 μl) of the PCR was separated on a 1% agarose gel. A singleband was noted and cloned into pGEM-T-Easy. The insert was sequenced andshown to contain most of the 5′ end of Tribdesat4 and the entire 3′ endof Tribdesat4. The existing 5′ end of Tribdesat4 was then subject toBLASTN analysis against available sequences of ESTs from T. castaneum.One EST was identified from this analysis (Accession No. DT795463) andthe rest of the 5′ end was identified.

Primers were designed to the 5′ and 3′ end of the coding region ofTribdesat4 and used in an RT-PCR reaction to obtain full lengthTribdesat4. The RT-PCR reaction consisted of 25 μl 2× Reaction mix(Invitrogen), 37 ng RNA, 1 μl Superscript II RT/Platinum Taq DNApolymerase (Invitrogen), 100 pmol of each primer and sterile water to 50μl. The reaction conditions were as follows: 50° C./30 min, 94° C./2min, then 35 cycles of 94° C./30 sec, 55° C./1 min, 72° C./1 min, then72° C./5 min. An aliquot was run on a 1% agarose gel. A single band(approximately 1 kb) was obtained and cloned into pGEM T Easy. Thesequence of the insert was examined and verified as the full lengthcDNA. SEQ ID NO: 6 shows the nucleotide sequence of the coding region ofthe cDNA.

Example 3 Amplification of Desaturase Sequences from Adult Tribolium RNA

RNA was extracted from T. castaneum (strain TC4) beetles using theTrizol method (Invitrogen). 100 mg of adult beetles were homogenized in1 ml Trizol reagent and the mixture incubated for 5 minutes at 25° C.,after which 200 μl chloroform was added to the sample. The mixture wasshaken by hand for 15 seconds and then allowed to settle for 3 minutesat 25° C. The organic and aqueous layers were separated bycentrifugation (12,000 g, 15 minutes, 4° C.). The upper aqueous layerwas transferred to a fresh tube and the RNA precipitated by the additionof 500 μl isopropanol. After 10 minute incubation at 25° C., the RNA waspelleted by centrifugation at 12,000 g/10 minutes at 4° C. The RNApellet was washed once with 75% ethanol and then air-dried for 10minutes. The RNA was then dissolved in 30 μl of RNase-free water. Therewas a noticeable brown colouration to the RNA which was further purifiedusing the RNeasy mini protocol for RNA cleanup (QIAgen), according tothe manufacturer's instructions.

RT-PCR amplification reactions were carried out for 10 desaturasesequences (Tribdesat1, 2a, 2b, 3, 5, 6a, 6b, 8, 10 and 11). To eachsample was added 25 μl 2× Reaction mix (Invitrogen), 37 ng RNA, 1 μlSuperscript II RT/Platinum Taq DNA polymerase (Invitrogen), 100 pmol ofeach primer and sterile water to 50 μl. The reaction conditions were asfollows: 50° C./30 min, 94° C./2 min, then 35 cycles of 94° C./30 sec,55° C./1 min, 72° C./1 min, then 72° C./5 min (PCR conditions 55/72).

An aliquot of each sample was run on a 1% agarose gel. Those samplescontaining a single amplification product of about 1 kb (Tribdesat1, 3,5, 6a, 10, 11) were purified using the QIAquick PCR purification kit(QIAgen) and cloned into pGEM T Easy (Promega). Ligation mixtures weretransformed into E. coli DH10B using standard molecular biologicaltechniques (Sambrook et al., 1989). The DNA sequence of clonescontaining inserts was examined. Comparisons of gene predictions withthose of RT-PCR products obtained for Tribdesat2b, 3, 6b, 10 and 11 areshown in FIGS. 1 to 5. It was apparent that the sequences as predictedfrom the genome sequence were not the same as the observed cDNAsequences, in some cases this would have been due to incorrectpredictions for coding sequences from the genomic sequence. In othercases, differences such as single nucleotide polymorphisms may have beendue to different alleles of the genes present in populations of theinsects.

RNA was also extracted from T. castaneum larvae using the Trizol methodas above. RT-PCR reactions were carried out to amplify 6 desaturasesequences (Tribdesat2a, 2b, 2c, 6b, 8 and 12) by the method see asabove. No amplification products were observed after a first round ofPCR and so the reactions were subject to a second round of PCR.Amplification products of about 1 kb were observed in the Tribdesat2b,Tribdesat2c, Tribdesat12 and Tribdesat8 samples. These products wereextracted from an agarose gel using the QIAquick gel extraction kit(QIAgen) and cloned into pGEM T Easy (Promega). The DNA sequences ofclones containing inserts were examined. Comparisons of gene predictionswith those of RT-PCR products obtained for Tribdesat2b and 6b are shownin FIGS. 1 and 3, respectively.

Example 4 Amplification of Desaturase Gene Fragments from SoldierBeetles (Chauliognathus lugubris)

Amplification of Internal Desaturase Fragments from Male Soldier Beetles

RNA was extracted from 100 mg of adult male C. lugubris beetles usingthe Trizol method as described above for Tribolium. The synthesis ofcDNA was performed using the Invitrogen Superscript II and using a polyTprimer (5′TTTTTTTTTTTTTTTTTT (SEQ ID NO: 81)). The polyT primer (100pmol), RNA (2 μl) and RNase-free water to a final volume of 15.5 μl wereincubated at 70° C. for 10 minutes and then chilled on ice. To this wasadded 10×PCR Buffer (2.5 μl), 25 mM MgCl₂ (2.5 μl), 10 mM dNTP mix (1μl) and 0.1M DTT (2.5 μl). This mixture was heated to 42° C. for 1minute after which Superscript II Reverse Transcriptase (1 μl) was addedand the mixture left for 50 minutes at 42° C. Superscript II ReverseTranscriptase was inactivated at 70° C. for 15 minutes and RNA degradedwith 1 μl RNaseH (2 Units, 30 minutes at 37° C.).

PCR reactions were setup containing 1 μl dNTPs (200 μM each dNTP), 5 μl10× ThermoPol Buffer (NEB), 1.5 μl F2 (5′TTCTTCTTCKCNCAYKTHGGNTGG (SEQID NO: 82)), 1.5 μl R2 (5′TGRTGGTAGTTGTGVHANCCCTC (SEQ ID NO: 83)), 5 μlcDNA; 0.1 U Taq DNA Polymerase (NEB) and sterile water to 50 μl. The PCRconditions were as follows: an initial denaturation of 94° C. for 3minutes and then 30 cycles of 94° C./15 s, 48° C./30 s, 72° C./2 min andthen a final extension of 72° C./5 min. An aliquot was separated on a1.5% agarose gel and a band of approximately 400 bp was visualised. ThePCR reaction was purified using the QIAgen QIAquick PCR purification kitaccording to the manufacturer's instructions and cloned into pGEM T Easy(Promgea). Approximately 30 clones containing inserts were examined byDNA sequence analysis. A total of three unique desaturase sequences wereidentified corresponding to CL6 (SEQ ID NO: 75), CL7 (SEQ ID NO: 76) andCL8 (SEQ ID NO: 77).

Amplification of Internal Desaturase Fragments from Female SoldierBeetles

RNA was extracted and cDNA synthesised as described above. Twodegenerate PCRs were performed on female adult RNA using the degenerateprimers F2/R2 and Clu_f (5′GCNCAYMGNYTNTGGGCNCA (SEQ ID NO: 84))/Clu-r(5′AANRYRTGRTGGTAGTTGIG (SEQ ID NO: 85)) (see Table 4) by the samemethod as above. Amplification products of approximately 400 bp (F2/R2)and 550 bp (Clu_f/Clu_r) were visualised and cloned into pGEM T Easy.Each insert from 70 colonies from the F2/R2 transformation was amplifiedby PCR using the T7 (5′TAATACGACTCACTATAGGG (SEQ ID NO: 86)) and SP6(5′ATTTAGGTGACACTATAG (SEQ ID NO: 87)) primers and analysed. Sinceapproximately 70% of the sequenced clones from male soldier beetletissue that had sequence similarity with desaturases were CL6, the PCRproducts were digested with restriction enzyme RsaI to remove CL6clones. Of the 70 PCR products obtained, only 20 were not digested withRsaI. Resultant desaturase fragments obtained were CL7, CL8 and a newunique desaturase fragment called CL9.

Sixteen further clones were obtained from another degenerate PCRreaction using female adult RNA and primers Clu_f/Clu_r, and theirnucleotide sequences obtained and compared to the previous clones. Oneof these was designated CL1.

TABLE 4 The tissue source of RNA and degenerate primer pairs used toisolate internal desaturase fragments from C. lugubris. Internaldesaturase Tissue Source Degenerate Primers products Male adult F2/R2CL6, CL7, CL8 Female adult F2/R2 CL9 Female adult Clu_f/Clu_r CL1 Mixedmale/female adult XRF2b/Clu_r CL3, CL5 defence gland

Amplification of Degenerate PCR from Defence Gland Tissue

A mixed population of males and females were dissected to obtain theirdefence gland tissue. Initially, the heads and the elytra were removed.The remaining tissue was divided into two parts—the abdomen sample andthe defence gland tissue.

RNA was extracted and cDNA synthesised as described above. DegeneratePCR was performed with three different combinations of primers. Thesewere Clu_f/Clu_r, F2/Clu_r and XRF2b (5′TTYTTYTWYKCNCAYATGGGNTGG (SEQ IDNO: 88))/Clu_r. After analysis on a 1.5% agarose gel, a band of theexpected size was observed only in the sample with the XRF2b/Clu_rcombination. No amplification products were observed with either of theother two pairs even after 60 cycles of PCR. The PCR products obtainedwere purified using the QIAgen QIAquick PCR purification kit and clonedinto pGEM-T-Easy. Eleven clones were examined by sequence analysis. Newdesaturase gene fragments were obtained and designated CL3 and CL5. TheCL5 internal fragment appeared to contain remnants of an intron, asdetermined by a section that did not show close sequence similarity withdesaturases and also affected the reading frame, thereby inserting astop codon.

5′ and 3′ RACE to Obtain Full Length Sequence Information

Primers for 5′- and 3′-RACE (Table 5) were designed based on sequencesinternal to the degenerate primer binding sites for each of the clonesdescribed above.

TABLE 5 Primers (5′ to 3′) used for 5′ and 3′ RACE. 5′RACE primer3′RACE primer Clone (CLXfront) (CLXend) CL6 CCCAGAGATATAACCGATACATTATATACGCCGACCCTATTC (SEQ ID NO: 89) (SEQ ID NO: 90) CL7CCTCTCCAAGTCCGAGAGAAG TGGCTCATGTGCAAAAAGCAT (SEQ ID NO: 91)(SEQ ID NO: 92) CL1 TTGTGCCATCCCTCACCGTTG TGGCTAACATCGAGACCCTG(SEQ ID NO: 93) (SEQ ID NO: 94) CL3 GCCATATAGATGAGCAGCTGAATGGGATGGCATTATGTGCAG (SEQ ID NO: 95) (SEQ ID NO: 96)

The Clontech Creator™ SMART™ System was used to obtain both 5′ and 3′RACE products according to the manufacturer's instructions with RNA fromeither abdomen or defence gland tissue. Oligonucleotide primers Smart IV(5′ AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG (SEQ ID NO: 97)) and CDSIII/3′ (5′ ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)₃₀N⁻¹N; N=A,G,C or T; N⁻¹=A,Gor C (SEQ ID NO: 98)) were used for the reverse transcription-PCR. The5′ RACE reactions used primer CDS V 5′AAGCAGTGGTATCAACGCAGAGT (SEQ IDNO: 99), and CLXfront primer. The 3′ RACE reactions used primers CDSIII/3′ PCR and CLXend.

Clones were screened by colony PCR for insertions. Any clones that gavea band of the expected size (˜350 bp) were examined by DNA sequenceanalysis. The DNA sequence was initially examined for sequencesimilarity with desaturases. Full length sequences were compiled insilico using sequence information from internal sequence, 5′RACE and3′RACE products and examined for an open reading frame beginning with anATG and ending in a stop codon that when translated possessed all themotifs of desaturases (Table 2).

Example 5 Isolation of Full Length C. lugubris Desaturase cDNAs

Full length cDNA clones of CL1 and other desaturase-like sequences wereobtained using a one-step RT-PCR kit from Invitrogen according to themanufacturer's instructions and PCR conditions 55/72 (above). Theprimers used were GGTACCATGCCACCCAACGCCCAC (SEQ ID NO: 100) andGAATTCTCACTTTTGTTTGTGAGT (SEQ ID NO: 101). Bands of approximately 1 kbwere purified using the QIAquick PCR purification kit from QIAgenaccording to the manufacturer's instructions and cloned intopGEM-T-Easy. Positive clones were obtained and examined by DNA sequenceanalysis. After verification of sequence, the pGEM-T-Easy clonescontaining the desaturase cDNAs were digested with restriction enzymesto excise the inserts, separated on a 1% agarose gel and the ˜1 kbfragments were excised and purified using the QIAquick gel extractionkit (QIAgen) for cloning into yeast expression vectors such as pYES2(below).

Example 6 Isolation of Desaturase Genes from Chauliognathus nobiliatus

Chauliognathus nobiliatus (Erichson) is another species of soldierbeetle, which resembles C. lugubris but is generally smaller in size andless prevalent in the south-east regions of Australia. These beetlespecies are not closely related to moths such as Tribolium, indeed theyare widely separated within the Insectae.

RNA was extracted from C. nobiliatus adults using the Trizol method asabove. cDNA was synthesised using SuperScript II kit from Invitrogen.Three degenerate PCRs were performed. The first used the degenerateprimers F2/Clu_r, the second used the degenerate primers XRF2b/Clu_r andthe third with degenerate primers Clu_f/Clu_r. An aliquot of eachreaction was separated on a 1.5% agarose gel and a band was observed forthe first two reactions. These were purified using the QIAquick PCRpurification kit (QIAgen) and cloned into pGEM-T-Easy. A total of 12clones from the first PCR and 14 from the second PCR were examined byDNA sequence analysis. Three unique desaturase sequences were obtainedand their corresponding orthologues from C. lugubris as well as thesignature motif as described by Knipple et al. (2002) are shown in Table6.

TABLE 6The internal desaturase fragments isolated from C. nobiliatus and theircorresponding orthologues in C. lugubris. Also shown is the signaturemotif and the primers used to isolate these internal fragments.C. nobiliatus C. lugubris Signature Primers CN8 CL8 GPAE F2/Clu_r(SEQ ID NO: 123) CN7 CL7 NPVE CL7end/CL7front (SEQ ID NO: 124) CN1 CL1YPAE CL1end/CL1front (SEQ ID NO: 125) CN9 CL9 SPVE CL9end/CL9front(SEQ ID NO: 126)

A second approach used to isolate internal desaturase fragments from C.nobiliatus was to use primers specific to C. lugubris desaturases. ThecDNA synthesised as described above was used as a template for PCR withspecific primers to CL6, CL7, CL9, and CL1. An aliquot of each reactionwas separated on a 1.5% agarose gel and bands were observed with theCL7, CL9 and CL1-specific primers but no products were observed foreither the CL6 or the CL10 primers. The CL7, CL9 and CL1 products werepurified using the QIAgen QIAquick PCR purification kit and cloned intopGEM-T-Easy. Positive clones were obtained and examined by DNA sequenceanalysis.

Full-length cDNA sequences for the CL3 and CL1 orthologues wereamplified from C. nobiliatus RNA using the One-step SuperScript IIReverse Transcriptase/Platinum Taq DNA Polymerase kit from Invitrogen asdescribed in Example 5. The primers used are shown in Table 7. A cloneobtained from each reaction was sequenced to confirm identity of eachcDNA.

TABLE 7 Primers used to obtain full length C. nobiliatusorthologues of selected desaturases. Forward Primer Reverse Primer Gene(5′→3′) (5′→3′) CL1 GGTACCATGCCACCCAACGC GAATTCTCACTTTTGTTTGTGACCAC (SEQ ID NO: 100) GT (SEQ ID NO: 101) CL3 AAGCTTATGCTACCGGAGTTCTCCAGCTAAGACTGATTATA TTGC (SEQ ID NO: 102) GGC (SEQ ID NO: 103)

Example 7 Isolation of Desaturase Genes from cDNA Libraries

Construction of a cDNA Library from C. lugubris Abdomen Tissue

RNA was extracted from C. lugubris abdomen tissue using the methoddescribed above. A cDNA library was constructed using the InvitrogenCloneminer kit according to the manufacturer's instructions. A fractionof the library was transformed into E. coli DH10B ElectroMax competentcells (Invitrogen) and transformants selected on LB agar platescontaining 25 μg/ml kanamycin. Approximately 90% of the clones containedinserts with an average insert size of 1.5 kb. Desaturase clones wereidentified and isolated from this library by PCR methods or byhybridisation screening using probes derived from the clones describedabove.

PCR Method for Screening Plasmid cDNA Library

A volume of plasmid cDNA library representing approximately 50 colonyforming units was seeded into 96 well plates containing 300 μl LBsupplemented with 50 μg/ml kanamycin and grown overnight at 37° C. withshaking. 100 μl of each liquid culture from the wells across the platewere pooled into 1.5 ml tubes to yield eight “lane pools” and a plasmidextraction was performed using a QIAquick miniprep spin kit (Qiagen)according to manufacturers instructions. 10 μl of each “lane pool”elutant was mixed into a 1.5 ml tube creating a “plate pool”. PCRscreening was done on each “plate pool” using gene specific primers andrun out on 1% TAE agarose gel to identify positive plate pool/s. Oncepositive plate/s were identified PCR screening was repeated on each“lane pool” corresponding to a positive “plate pool” and run out on 1%TAE agarose gel to identify positive “lane pools”, and in subsequentscreening rounds on single wells and individual colonies to isolatepositive clones. Plasmids which contained the gene of interest wereanalysed by restriction enzyme digest and sequence analysis.

Screening Using Non-Radioactive Colony Hybridisation.

10-fold serial dilutions of the cDNA library were made (1-10⁶) andplated on LB agar supplemented with 50 μl/ml kanamycin to determine thedilution which produced approximately 300-500 colonies per 82 mm petridish. Transformed bacteria of a suitable dilution were spread on LB agarplates supplemented with 50 μg/ml kanamycin and incubated overnight at37° C. Nylon membranes (Hybond-N+, Amersham Biosciences) were used toobtain colony lifts and treated according to standard methods for colonyhybridisation. Biotin labelled DNA probes were prepared using desaturasegene fragments labelled using dNTPs plus Biotin-dATP and DNA polymeraseI Klenow fragment according to NEBlot phototope kit (New EnglandBiolabs). Probes were hybridised to the membranes overnight attemperatures of 65° C. (high stringency) or 55° C. (moderatestringency). The hybridised biotinylated DNA was detected according tothe Phototope-Star detection Kit manual (New England Biolabs) followingthe modification for colony hybridisations detailed in Appendix C. Themembrane was exposed to Hyperfilm ECL (Amersham Biosciences) for 1minute and processed manually according to Amersham Biosciencesrecommendations. Positively hybridising clones were analysed byrestriction enzyme digest and sequence analysis.

Example 8 Yeast Expression Vector Construction and Functional Analysisof Genes

Each of the full-length desaturase genes was expressed in Saccharomycescerevisiae for functional characterization using the yeast expressionvectors pYES2 or pVT100-U (Vernet, T et al. 1987). Each of the genomicregions containing putative desaturase genes was examined forrestriction nuclease recognition sites. Restriction enzymes were chosenthat did not cut the genomic regions but would facilitate directionalcloning into pYES2 or pVT100-U. Primers were designed (Table 8) to themost 5′ and 3′ regions of the predicted desaturase genes withrestriction sites at the 5′ end of each of the primers to allowdirectional cloning.

DNAs of the yeast vectors were prepared and digested with thecorresponding restriction enzymes and then dephosphorylated using CalfIntestinal Alkaline Phosphatase (Roche). After dephosphorylation, thedigested vectors were purified using the QIAquick PCR purification kit(QIAgen). Gel extracted desaturase-containing fragments were ligatedwith digested vector and ligations transformed into E. coli DH10B withtransformants selected on LB agar plates with 100 μg/ml ampicillin.Clones containing inserts were confirmed by restriction enzyme analysis.

TABLE 8 Primers used for directional cloning into pYES2.Restriction sites are shown in bold face. Tribdesat No. PrimerOligonucleotide primer sequence  1 Forward GGATCCATGGCCCCCAACAGCACA(SEQ ID NO: 37)  1 Reverse GAATTCTTAATCTCTGCGTGTGCG (SEQ ID NO: 38)   2aForward GGATCCATGTCAACGCTTGAAACA (SEQ ID NO: 39)   2a ReverseGAATTCTTATCCTCGATTTCGTTC (SEQ ID NO: 40)   2b ForwardGGATCCATGTCTAGCGAGCTAGCG (SEQ ID NO: 41)   2b ReverseGAATTCTTAATTTTTCGCCTTACA (SEQ ID NO: 42)   2c ForwardGGATCCATGGAACGTGAAATCGCGTGG (SEQ ID NO: 43)   2c ReverseGAATTCTTATCCTGTTTGTGAAGC (SEQ ID NO: 44)  3 ForwardGGATCCATGTTTTTACGTACAATA (SEQ ID NO: 45)  3 ReverseGAATTCTTAATAATCACAATCCCC (SEQ ID NO: 46)  4 ForwardAAGCTTATGACGGAAGGCAGCGATGAA (SEQ ID NO: 47)  4 ReverseGCGGCCGCTCAGTTAAAATCCTCCATTTT (SEQ ID NO: 48)  5 ForwardGGATCCATGCCACCCTATGTGTCC (SEQ ID NO: 49)  5 ReverseGCATGCTTAATTAAAATCGTCAGA (SEQ ID NO: 50)   6a ForwardGGATCCATGACACCAAATGCTTCA (SEQ ID NO: 51)   6a ReverseGAATTCCTACGCACTCTTCCTATG (SEQ ID NO: 52)   6b ForwardGGTACCATGCTAATCTTACTTTCC (SEQ ID NO: 53)   6b ReverseCTCGAGCTAATGAAATTTTGAAGG (SEQ ID NO: 54)   7a ForwardGGATCCATGTTTCAAACACCCATCGTCTGG (SEQ ID NO: 55)   7a ReverseCTCGAGTTATTGCCCAGTCCTCAAAACCCGCTT (SEQ ID NO: 56)   7b ForwardGGATCCATGGTAGATTTGTTTTTG (SEQ ID NO: 57)   7b ReverseGAATTCTTATTTTTGCAATTGTTT (SEQ ID NO: 58)  8 ForwardGGATCCATGGCTCCAAATTCGCTC (SEQ ID NO: 59)  8 ReverseGAATTCTTACAGTTCTTTGCTACT (SEQ ID NO: 60) 10 ForwardGGATCCATGTCGGCCCAGACCATT (SEQ ID NO: 61) 10 ReverseGAATTCTTAATCCTCCTTCCTGTT (SEQ ID NO: 62) 11 ForwardAAGCTTATGGGAGCGCTCAAACAA (SEQ ID NO: 63) 11 ReverseGAATTCTTAACCATTTGCCGTAAC (SEQ ID NO: 64) 12 ForwardGAATTCATGGCTCCTAATTTGCTAGGA (SEQ ID NO: 65) 12 ReverseCTCGAGTTAATCAAATTTCTCTCTACT (SEQ ID NO: 66)Transformation of Expression Constructs into Saccharomyces cerevisiae

Two S. cerevisiae host strains were used as recipients for pYES-derivedconstructs. These were S288C (genotype MATα, SUC2 gal2 mal mel flo1flo8-1 hap1 (Mortimer and Johnston (1986)) and OLE1 (his3Δ1, leu2Δ0,ura3Δ0, YMR272c::kanMX4) which is mutant for the gene encodingΔ9-desaturase and could be used for complementation analysis. Five S.cerevisiae host strains were used as recipients for pVT-100 derivedconstructs. These were S288C, OLE1, and INVSCi (MATa, his3D1 leu2trp1-289 ura3-52), YPH499 (MATa, ura3-52 lys2-801_amber ade2-101_ochretrp1-Δ63 his1-Δ200 leu2-Δ1) and ELO-1 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0ΔELO1). These strains were treated the same during transformation exceptthat 17:1 (cis-10-heptadecenoic acid, 1 mM) and tergitol (NP-40; 1%)were added in all media in which S. cerevisiae OLE1 was grown andgeneticin (200 μg/ml) was added to all media in which ELO-1 was grown.

For transformation, each S. cerevisiae strain was streaked onto YPD (20g/l peptone, 10 g/l yeast extract, 2% glucose) and grown for severaldays at 30° C. Transformations were performed using the Sigma YeastTransformation kit according to manufacturer's instructions.Transformants were selected on SCMM-U agar plates (6.7 g/l yeastnitrogen base without amino acids, 1.92 g/l yeast synthetic drop-outmedia supplement without uracil, 2% glucose and 2% agar) at 30° C. forup to 5 days. A number of transformants were selected for each constructand tested for the presence of plasmid DNA.

Confirmation of Transformants

To confirm the identity of the expression constructs withintransformants, DNA was isolated from a loopful of growth using the Y-DERyeast DNA extraction kit from Pierce (Ill.) according to themanufacturer's instructions. The presence of the plasmid was analysedand confirmed by PCR analysis using a gene specific reverse primer and aT7 primer (vector primer). Positive bands were observed and thecorresponding transformants were retained for further analysis.

Assessment of OLE1 Complementation

Fifty microlitres of OLE1 transformants grown on SCMM-U containingcis-10-heptadecenoic acid (1 mM) and 1% tergitol was pelleted bycentrifugation (13,000 rpm, 3 ses) and resuspended in 50 μl YP (20 g/lpeptone, 10 g/l yeast extract). The resuspended mixture was diluted 1:10and 20 μl of each mixture was spotted onto YP containing 2% galactoseplates to induce desaturase gene expression and these were incubated for3-5 days at 30° C. The presence or absence of growth was noted. Controlplates containing cis-10-heptadecenoic acid (1 mM) and 1% tergitol werealso used to ensure yeast viability. The results for the OLE1complementation tests for Tribolium and Chaulioganthus desaturases wereas follows. Positive complementation was observed for Tribdesat2a,Tribdesat5, Tribdesat6a and Tribdesat11, the last one with a lag phase.Possibly weak complementation was observed for Tribdesat2b andTribdesat8. No complementation was observed for Tribdesat1, Tribdesat2c,Tribdesat4, Tribdesat10, Tribdesat12, CL1 and CL3.

Complementation of the yeast OLE1 phenotype occurs when a heterologouslyexpressed gene expresses a functional Δ9- or Δ11-desaturase and resultsin the production of palmitoleic, oleic, or cis-11-octadecanoic fattyacids. Therefore, it was concluded that Tribdesat2a, Tribdesat5,Tribdesat6a and Tribdesat11 were desaturases able to desaturate thenative lipids of OLE1 yeast cells to produce one of the complementingfatty acids and therefore were likely to be Δ9 desaturases active onC16:0 or C18:0, or All desaturases active on C18:0, whereas Tribdesat1,Tribdesat2c, Tribdesat4, Tribdesat10, Tribdesat12, CL1 and CL3 wereunlikely to encode one of these activities and therefore likely toencode a different desaturase.

Growth of Transformed S. Cerevisiae for Substrate Feeding Experiments

Yeast transformants derived from stains S288C or OLE1 were inoculatedinto 25 ml of synthetic minimal defined medium for yeast without Uracil(SCMM-U medium) containing 2% glucose and additionally 0.5 mMcis-11-heptadecenoic acid for the OLE1 strain. This inoculation culturewas grown for 24-48 hr at 30° C. with shaking. The OD600 of the culturewas determined. From this, the amount of culture necessary to obtain anOD600 of 0.4 in 50 ml of induction medium was calculated. This amount ofinoculation culture was removed and the cells pelleted at 1500×g for 5minutes at 4° C. The cells were then resuspended in 10 ml of inductionmedium, SCMM-U containing 2% galactose, 1% raffinose and 0.5 mM fattyacid substrate (added in ethanol/20% tergitol) and additionally 0.5 mMcis-11-heptadecenoic acid for OLE1 derived transformants. Each culturewas grown for 24-48 h at 30° C. with shaking. Either 2 ml or the totalculture was then used for analysis of fatty acids after harvesting cellsby centrifugation, storage at −20° C. if needed.

Extraction of Lipids from Transformed S. cerevisiae and Analysis

The yeast cells from fatty acid feeding experiments were washed in 1%tergitol in water and pelleted at 1500×g for 5 minutes at 4° C. Cellpellets (200-500 mg) were then resuspended in water, transferred toglass test tubes and the cells again pelleted. Water was removed fromthe cell pellets in a Savant SpeedVac Plus SC110A concentrator/dryer.Each pellet was used either used directly or lipids were extracted withsolvent as described below.

Lipids were extracted based on a Modified Folch Method (Protocol 7, pp22-24, Lipid Analysis, Hamilton and Hamilton, 1992). Lipids wereextracted with 2 ml chloroform/methanol (2:1), and 0.5 ml of a salinesolution, 0.9% w/v NaCl in water, was added. This was mixed thoroughlyby vortexing. The layers were then allowed to separate; when a largeamount of cell debris was present the samples were centrifuged at 2500×gfor 5 minutes. The top aqueous layer was removed and discarded. Thebottom solvent layer was removed to a clean glass test tube. This wasthen dried down under nitrogen at 30° C.

Lipids were then derivatised prior to analysis by two differentmethylation methods. The first was the basic methylation of incorporatedfatty acids, i.e. those found in triacyl glycerides or phospholipids,not free fatty acids, based on the method of Christie (2003). The lipidsample was first dissolved in 0.5 ml hexane. Methyl acetate, 60 μl, wasadded, followed by 50 μl sodium methoxide at 0.5 M in methanol. Thesealed test tube was then mixed thoroughly and placed in a heating blockat 50° C. for 10 minutes. After being allowed to cool for 5 minutes, adrop of acetic acid was added. The sample was dried in a gentle streamof nitrogen at 30° C., dissolved in 200 μl hexane and transferred to avial prior to GC analysis.

The second method was an acidic methylation of all free and incorporatedfatty acids, based on the method of Lewis et al. (2000). A methanolicsolution, 2 ml of methanol/hydrochloric acid/chloroform (10:1:1), wasadded to the dried yeast pellet or extracted lipid sample. The sealedtest tube was mixed thoroughly and placed in a heating block at 90° C.for 60 minutes. After being allowed to cool for 10 minutes, 1 ml of 0.9%saline (NaCl in water) was added. The methylated fatty acids were thenextracted with 0.3 ml hexane. The hexane layer was transferred to a vialprior to analysis.

These fatty acid methyl ester (FAME) samples were analysed by GC andGCMS. GC analysis was carried out with a Varian 3800 gas chromatographfitted with a flame ionisation detector (FID) and a BPX70 capillarycolumn (length 30 m, i.d. 0.32 mm, film thickness 0.25 μm). Injectionswere made in the split mode using helium as the carrier gas and aninitial column temperature of 100° C. The temperature was raised at 3°C./minute until 150° C., then raised at 5° C./minute until 170° C., heldfor 5 minutes, then raised at 50° C./minute until 255° C. GC/MS analysiswas carried out under similar chromatography conditions but with aninitial column temperature of 60° C., raised at 20° C./minute until 170°C., held for 5 minutes, then raised at 50° C./minute until 255° C.Detection was carried out using either a TEC PolarisQ Ion Trap or aVarian 1200 Single Quadrupole mass spectrometer. Mass spectra wereacquired under positive electron impact in full scan mode between 50-400amu at the rate of 2 scans per sec. The mass spectra corresponding toeach peak in the chromatogram was automatically compared with spectra inthe computerised NIST library. Test spectra that matched library spectrawith a high degree of accuracy and eluted at the same time as anauthentic standard or eluted at a plausible retention time, weretentatively identified. Confirmation of the identity of a fatty acid wasachieved by the conversion of the fatty acid to its dimethyloxazoline(DMOX) derivative using the method of Yu et al. (1988) and comparisonwith DMOX mass spectra described in Dobson and Christie (2002) andreferences within.

Results of Functional Analysis in Yeast.

From the experiments carried out as described above and summarised inTable 10 (below), it was concluded as follows:

Tribdesat2a had Δ9 desaturase activity on saturated fatty acid carbonchain length from C10:0 to C16:0 when these fatty acids were fed to theyeast transformants. Activity was greatest on 14:0, but 15:0 and 16:0were also efficient substrates. It had no detectable desaturase activityon 18:0, 20:0 or even longer saturated fatty acids in the yeast cells.The protein had a predicted size of 320 amino acids and included the 8conserved desaturase motifs and three His boxes (Table 3). On the basisof the sequence homology with known acyl-CoA desaturases, this enzymewas presumed to be acting on the acyl-CoA substrate. This enzyme wastherefore characterised as a myristoyl-CoA Δ9 desaturase.

Tribdesat2b and Tribdesat2c were closely related proteins, having 64%amino acid sequence identity based on global alignment using BLOSUM62,which catalysed production of 5-hexadecenoic and 5-octadecenoic acidsfrom palmitic acid and stearic acid, respectively. They were thereforecharacterised as Δ5 desaturases acting on the saturated substrates C16:0and C18:0. The efficiency of conversion in yeast cells of C18:0 toC18:1^(Δ5) was more than twice as efficient as the conversion of C16:0to C16:1^(Δ5) (Table 9). The enzymes were able to convert at least 8.0%of the substrate to the product in each case, to at least 40% forTribdesat2b on C18:0. The enzymes did not detectably desaturatesubstrates of C14 or shorter when fed to the yeast cells, includingC14:0 or C14:1^(Δ9), or the substrates of C20 or longer including C20:0or C20:1^(Δ13). Furthermore, the enzymes did not desaturatemonounsaturated or polyunsaturated fatty acids including C16:1Δ9,C18:1Δ9, C18:2 or C18:3. The proteins included the 8 conserveddesaturase motifs and three His boxes (Table 3) and showed sequencehomology with known acyl-CoA desaturases. They were thereforecharacterised as stearoyl-CoA Δ5 desaturases. They also havepalmitoyl-CoA Δ5 desaturase activity.

TABLE 9 Fatty acid composition (% total fatty acids) of yeast cellsexpressing Tribdesat2b, or empty pYes vector, supplied with 0.5 mM 18:0.Fatty acid composition (% total fatty acids) Beetle desaturase 12:0 14:015:0 16:0 16:1Δ5 17:0 17:1 18:0 18:1Δ5 TD2b + 18:0 2.4 0.7 0.5 24.3 3.20.2 62.4 3.2 3.0 pYes empty + 18:0 1.6 0.4 0.4 22.7 0 0.4 65.7 8.9 0

Cahoon et al. (2000) isolated a gene from Limnanthes (meadowfoam) whichencoded a Δ5 desaturase which was more active on C20:0 than C18:0 orC16:0 and is therefore was not a stearoyl-CoA Δ5 desaturase as definedherein. Sayanova et al. (2007) cloned two genes encoding Δ5 desaturasesactive on acyl-CoA substrates from Anemone leveillei seeds. Thedesaturase AL21 was active on both saturated and unsaturated substrates,C16:0, C18:0 and C20:2, while AL10 was active only on C20:2, n-6.However, AL21 was more active on the unsaturated substrate than thesaturated substrate so it would not be considered as a stearoyl-CoA Δ5desaturase as defined herein. The degree of identity in amino acidsequences when the proteins were compared over the entire sequenceswere: between AL21 and Tribdesat2b, 25%; AL21 and Tribdesat2b, 27%; AL10and Tribdesat2b, 24%; AL10 and Trib2c, 28%. Therefore, it was concludedthat there was insignificant or little homology between the plantacyl-CoA dependent desaturases and the insect ones.

Tribdesat5 and 6a showed activity in yeast cells as Δ9 desaturases thatacted primarily and very efficiently on stearic acid to produce oleicacid. They also had some activity on palmitic acid to producepalmitoleic acid. The enzymes did not detectably desaturate substratesof C14 or shorter when fed to the yeast cells, including C14:0 orC14:1^(Δ9), or the substrates of C20 or longer including C20:0. Theywere therefore characterised as stearoyl-CoA Δ9 desaturases.

Tribdesat8 was able to desaturate palmitic acid to produce palmitoleicacid, so it was considered to be a Δ9 desaturase. It did not havedetectable desaturase activity in the yeast cells on C14 or shortersubstrates, or on C18:0 or longer saturated substrates, or onmonounsaturated or polyunsaturated substrates including C16:0^(Δ9),C18:1^(Δ9), C18:2 or C18:3. It therefore appeared to be specific forpalmitic acid. The protein had a predicted size of 374 amino acids andincluded the 8 conserved desaturase motifs and three His boxes (Table3). On the basis of the sequence homology with known acyl-CoAdesaturases, this enzyme was presumed to be acting on the acyl-CoAsubstrate. This enzyme was therefore characterised as a palmitoyl-CoA Δ9desaturase.

Tribdesat10 showed Δ12 desaturase activity on palmitoleic and oleicacids resulting in C16:2^(Δ9,Δ12) and C18:2^(Δ9,Δ12) (LA), respectively.Conversion of oleic acid substrate (at least 30% converted) was abouttwice as efficient in yeast cells compared to conversion of palmitoleicacid. There was no detectable activity on C16:0, C18:0 or longersaturated substrates, or C14 or shorter substrates including C14:0^(Δ9),so the enzyme appeared to be specific as a Δ12 desaturase acting onΔ9-mono-unsaturated substrates of C18 and C16 length joined to CoA. Theprotein had a predicted size of 366 amino acids and included the 8conserved desaturase motifs and three His boxes (Table 3). On the basisof the sequence homology with known acyl-CoA desaturases, this enzymewas presumed to be acting on acyl-CoA substrates. This enzyme wastherefore characterised as an oleoyl-CoA Δ12 desaturase. It also showedpalmitoleoyl-CoA Δ12 desaturase activity. It was believed this is thefirst gene to be characterised encoding such an enzyme.

Tribdesat11 showed Δ9 desaturase activity on saturated substrates havinga chain length from C14:0 to C24:0, with the greatest efficiency ofconversion observed for C22:0 (known as behenic or docosanoic acid) andC24:0 (known as lignoceric or teracosanoic acid). Therefore, this enzymewas considered to be a lignoceroyl-CoA Δ9 desaturase, although it alsohas behenoyl-CoA Δ9 desaturase activity.

CL1 showed Δ12 desaturase activity on palmitoleic and oleic acidsresulting in C16:2^(Δ9,Δ12) and LA, respectively, the same spectrum ofactivity as Tribdesat10 although the activity was less efficient thanfor Tribdesat10.

CL3 showed efficient desaturation activity on C14:0 to produceC14:0^(Δ9). The enzyme did not have detectable activity for C12:0 orC16:0 and therefore appeared to be a specific myristoyl-CoA Δ9desaturase. It did not have activity on mono-unsaturated substrates suchas C12:1Δ5 or C20:1. On the basis of the sequence homology with knownacyl-CoA desaturases, this enzyme was presumed to be acting on theacyl-CoA substrate. This enzyme was therefore characterised as amyristoyl-CoA Δ9 desaturase.

TABLE 10 Substrates (n.d. - no product detected) Desaturase 10:0 12:012:1Δ5 14:0 14:1Δ9 15:0 16:0 16:1Δ9 Tribdesat 2a 10:1Δ9 12:1Δ9 — 14:1Δ9— 15:1Δ9 16:1Δ9 — 0.5% 1.8% 48.6% 28.0% 27.8% Tribdesat 2b n.d. n.d. —n.d. n.d. — 16:1Δ5 n.d. 17.0% Tribdesat 2c n.d. n.d. — n.d. n.d. —16:1Δ5 n.d. 8.0% Tribdesat 4 n.d. n.d. — n.d. n.d. — n.d. n.d. Tribdesat5 n.d. n.d. — n.d. n.d. — 16:1Δ9 n.d. 11.8% Tribdesat 6a n.d. n.d. —n.d. n.d. — 16:1Δ9 n.d. 8.9% Tribdesat 8 n.d. n.d. — n.d. n.d. — 16:1Δ9n.d. 5.0% Tribdesat 10 n.d. n.d. — n.d. n.d. — n.d. 16:2Δ9 Δ12 15.9%Tribdesat 11 n.d. n.d. — 14:1Δ9 — n.d. 16:1Δ9 — 12.0% 8.3% Tribdesat 12n.d. n.d. — n.d. n.d. — n.d. n.d. CL1 n.d. n.d. n.d. n.d. n.d. — n.d.16:2Δ9 Δ12 0.1% CL3 n.d. n.d. n.d. 14:1Δ9 n.d. — n.d. n.d. 61.2%Substrates (n.d. - no product detected) Desaturase 18:0 18:1Δ9 18:2 18:320:0 20:1Δ13 22:0 24:0 Tribdesat 2a n.d. — — n.d. — n.d. n.d. Tribdesat2b 18:1Δ5 n.d. n.d. n.d. n.d. n.d. n.d. n.d. 43.3% Tribdesat 2c 18:1Δ5n.d. n.d. — n.d. n.d. n.d. n.d. 19.0% Tribdesat 4 n.d. n.d. n.d. n.d.n.d. — n.d. n.d. Tribdesat 5 18:1Δ9 n.d. n.d. — n.d. — n.d. n.d. 88.7%Tribdesat 6a 18:1Δ9 n.d. n.d. — n.d. — n.d. n.d. 79.2% Tribdesat 8 n.d.n.d. n.d. — n.d. — n.d. n.d. Tribdesat 10 n.d. 18:2Δ9 n.d. — n.d. — n.d.n.d. Δ12 30.5% Tribdesat 11 18:1Δ9 — n.d. — 20:1Δ9 — 22:1Δ9 24:1Δ9 35.6%14.7% 50.3% 58.3% Tribdesat 12 n.d. n.d. n.d. n.d. n.d. — n.d. n.d. CL1n.d. 18:2Δ9 n.d. n.d. n.d. n.d. n.d. n.d. Δ12 1.5% CL3 n.d. n.d. — —n.d. n.d. n.d. n.d.

Example 9 Cloning of Desaturase Genes into Gateway Entry Vector pENTR11

To construct the functional genes or genes to be tested in a Gatewayentry vector, the pGem-T-Easy constructs containing full-length genesequences were digested with restriction enzymes to excise the inserts.These were separated on a 1% agarose gel and the DNA of theapproximately 1 kb fragments purified using the QIAquick Gel Extractionkit (Qiagen) according to the manufacturer's instructions. The Gatewayentry vector pENTR 11 (Invitrogen) was prepared and digested with therequired restriction enzymes and dephosphorylated using Calf IntestineAlkaline Phosphatase (Roche). Gene fragments were ligated with thevector and transformed into E. coli strain JM109. Transformants wereselected on LB agar plates containing 50 μg/ml kanamycin. Clonescontaining inserts were confirmed by restriction enzyme and sequenceanalysis.

Cloning of Desaturase Genes into Gateway Destination Vectors to CreateExpression Clones.

Two expression system, SF9 insect cells and Arabidopsis, were selectedand Gateway expression vectors chosen for each. For SF9 insect cells,pDEST8 was chosen and selection for DH10Bac transformants carried out inLB supplemented with kanamycin (50 μg/ml), gentamicin (7 μg/ml) andtetracycline (10 μg/ml), Bluo-gal (100 μg/ml) and IPTG (40 μg/ml). ForArabidopsis transformation, binary vector pXZP391 (CSIRO Plant Industry)was chosen and selection for transformants carried out in LBsupplemented with spectinomycin (50 μg/ml).

pENTR11 clones containing the gene inserts and appropriate pDEST vectorswere prepared and under-went an LR recombination reaction according tomanufacturers instructions (Gateway LR clonase enzyme mix, Invitrogen).The resultant recombination mixtures were transformed into E. colistrain JM109 for pXZP391 or DH10Bac (Invitrogen) for pDEST8.Transformants containing correct inserts were confirmed by restrictionenzyme analysis and sequence analysis using vector specific primers.

Example 10 Recombinant Baculovirus Production of Insect Desaturases orAcetylenases

The Tribdesat2b and CL3 genes were cloned into the pDest8 vector(Invitrogen), using Gateway recombination as above. Competent DH10Baccells (Invitrogen) were transformed with the pDest8 clones by heat shockmethod. Transformants were selected on LB agar containing kanamycin,tetracycline and gentamycin (50 μg/mL, 10 μg/mL and 7 μg/mLrespectively) and IPTG and Xgal at the appropriate concentrations, andpurified by re-streaking on this medium. Once the recombination eventshad taken place in DH10Bac, the recombinant bacmids (now calledpFastbac) were identified using M13 forward and reverse primers tocolony screen the 2b and CL2 inserts in pFastbac in DH10Bac cells.Positive clones produced a band of approximately 3 kb. Clones werepicked for downstream applications that did not have any evidence ofempty vector contamination which could be observed as a 300 bp product.

Transformants containing pFastbac plasmids having the 2b and CL2 insertswere grown overnight in LB medium containing the antibiotics. A modifiedalkaline lysis plasmid isolation protocol was used according to themanufacturer's instructions in the Bac-to-Bac Expression System manual.Isolated DNA was dissolved in 40 μL of 1×TE buffer pH 8.0 andquantitated using the Nanodrop spectrophotometer and stored at 4° C.

Transfecting Insect Cells

Sf9 (Spodopetera frugiperda ovary cell line) cells were seeded in a 6well tissue culture plate at a rate of 9×10⁵ cells per well, in 2 mL ofSF900II serum free medium (Invitrogen). The cells were allowed to adhereto the well surface for 2 hours at 27° C. For each transfection, 1 μg ofpurified bacmid DNA was diluted in 100 uL of unsupplemented Grace'sMedium (Invitrogen), in polystyrene tubes. 6 μL of Cellfectin® Reagent(Invitrogen) was diluted in 100 μL of unsupplemented Grace's Medium inpolystyrene tubes. The DNA and Cellfectin® Reagent were then mixedtogether and incubated at RT for 30 minutes. While DNA:lipid complexeswere incubating, the cells were washed once with 2 mL of unsupplementedGrace's Medium and the medium was then removed. 0.8 mL of unsupplementedGrace's Medium was added to the DNA:lipid complex, mixed and then addedto the washed SF9 cells. The transfection was allowed to proceed at 27°C. for 5 hours. The DNA:lipid complexes were removed from the cells and2 mL of SF900II were added. Cells were incubated at 27° C. until signsof infection were obvious. Once the cells appeared infected, the viruswas harvested by centrifuging the cell culture medium at 1 Krpm for 5minutes at 20° C. The clarified cell culture medium was transferred totubes and stored at 4° C. This was called P1 viral stock.

Amplifying the Baculovirus Stocks

Sf9 cells were used to seed 25 cm² flasks at a rate of 1×10⁶ cells/mL in5 mL of medium. The cells were infected with P1 viral stock at amultiplicity of infection (MOI) of 0.1 virus particles per cell, whichwas estimated to be 5×10⁶ cells/mL. After 48 hours at 27° C., the cellswere showing signs of infection and the cell culture medium washarvested as outlined above. This was called P2 viral stock. The P2stock was then used to produce a P3 stock. 75 cm² flasks were seeded ata rate of 1×10⁶ cells/mL in 15 mL SF900II medium, and infected at a MOIof 0.1 virus particles per cell, which was estimated to be 5×10⁶cells/mL. After 48 hours at 27° C., the cells were showing signs ofinfection and the cell culture medium was harvested as outlined above.This was called P3 viral stock.

To determine the exact titre of each P3 stock, a TCID₅₀ (Tissue CultureInfectious dose) was performed. A 96 well tray was seeded with 3×10³cells per well.

Cells were incubated at 27° C. overnight. 10-fold dilutions of the viruswere prepared to 10⁻⁸. 50 μL of dilutions 10⁻³-10⁻⁸ were added to 8wells each. The TCID₅₀ was then incubated for 7 days at 27° C. The wellswere scored positive or negative for virus infection. Once the viruseswere titrated, infections of SF9 cells with known multiplicity ofinfections (MOI) could proceed. Infested cells were removed and driedunder vacuum.

Methyl esters of fatty acids of insect cells infected with the constructexpressing Tribdesat2b were obtained using the direct acidic methanolreagent as previously described and analysed by GC/MS. The GC/MS traceand spectrum showed C16:1Δ5 and C18:1Δ5 production by SF9 expressingTribdesat2b, as compared to the SF9 control. The efficiency ofconversion of C16:0 to C16:1d5 in the insect cells transduced with theconstruct was 16.9% and the efficiency of conversion of C18:0 to C18:1d5was 29.7%. These data therefore confirmed the data obtained from yeastcells expressing Tribdesat2b and it was concluded that this gene encodeda Δ5-desaturase active on C16:0 and C18:0 substrates, with greateractivity on the latter. It therefore almost certainly encoded aStearoyl-CoA Δ5-desaturase, which activity has not previously beenreported for a cloned gene. The other desaturases are likewise expectedto show activity in insect cells.

Example 11 Expression of Tribolium and Chauliognathus Genes in Plants

The Tribolium Tribdesat10 and 2b genes were cloned into pENTR11 asdescribed above and then recombined into plant expression vector pXZP391which provides for expression of the coding region insert under thecontrol of a seed-specific napin promoter (Fp1) isolated from Brassicanapus (Stalberg et al., 1993). The expression plasmid was transformedinto Agrobacterium tumefaciens AGL1, and used for plant transformationinto the Arabidopsis thaliana fad2/fae1 double mutant which lacksΔ12-desaturase activity, by in planta method. Seeds of spray-inoculatedplants were harvested 3-4 weeks later and plated onto selective media.T1 transformed plants were identified on MS media containing 40 mg/Lkanamycin and 100 mg/L timentin, and transferred into pots in theglasshouse. T2 seeds from each T1 plant were analysed for fatty acidcomposition by gas chromatography. The data shown in FIG. 6 demonstratedthe Δ12 desaturase activity of Tribdesat 10 in Arabidopsis, as LA wasproduced from oleic acid in the fad2/fae1 mutant plant.

Expression of Transgenes in Tobacco Calli and Plants

The Tribdesat 2b, CL1 and CL2 coding regions were cloned in the ‘sense’orientation into the plant expression vector pVEC8 (Wang et al., 1997)under the control of the regulatory sequences CaMV 35S promoter andoctopine synthase transcription termination/polyadenylation signal(Gleave, 1992), providing strong constitutive expression of thetransgene in most plant tissues. The transgene was introduced intotobacco by Agrobacterium-mediated transformation of leaf tissue (Horschet al., 1985) with selection of the transgenic cell lines onhygromycin-containing media. Transgenic plant cells were selected basedon the resistance to hygromycin, as conferred by a hpt resistance markergene in the pVEC8 derived vector. Transformed tobacco lines werecultured to produce either calli or regenerated to produce whole plants,depending on the phytohormone regime in the selection and growth media(Murashige and Skoog, 1962; Horsch et al., 1985). Undifferentiatedtransgenic tobacco calli were produced by selection on calli-inducingphytohormones (0.5 mg/L indole acetic acid (IAA) and 0.05 mg/L kinetin)and calli were maintained on agar plates. Differentiated tobacco plantswere produced by first selecting transgenic material on shoot-inducingmedia by the addition of shoot-inducing phytohormones (100 μg/LN-6-benzyladinine (BAP) and 500 ng/L L IAA). After 3 weeks of growth onthis media individual tobacco apices were cut from the leaf discs andre-plated onto root-inducing media containing root-inducingphytohormones (50 ng/L IAA). A further 3 weeks are required for theformation of roots. Transgenic material, either calli or whole plants,were selected for analysis as required.

The Tribdesat2b gene was also recombined using LR clonase into plantexpression vector pXZP393 (CSIRO Plant Industry) under control of theconstitutive CaMV 35S promoter. The resulting expression plasmid pXZP384was transformed into AGL1, and used for plant transformation intotobacco leaves as described above. Fatty acid composition of transgenictobacco leaves and seed will be analysed by GC and GC-MS to shownmodification of fatty acid composition by addition of the new desaturaseactivity.

Example 12 Cloning of Desaturase Genes from Acheta domesticus

Some insects are thought to possess Δ12-desaturase activity but as yetno gene encoding such an enzyme has been isolated despite extensiveeffort over 20 years. The reasons for this failure were unknown untilthe present work. One insect known to possess Δ12-desaturase activity isAcheta domesticus, the common cricket. In an initial attempt to clone anA. domesticus Δ12-desaturase gene, a set of degenerate primersAdD12Des-F1 (5′-TTGTTCTGTGTGGGTCAYGAYTGYGGWCA (SEQ ID NO: 127)) andAdD12Des-R1 (5′-GTGATGGGCGACGTGACYGTYKGTRAT (SEQ ID NO: 128) weredesigned based on the conserved histidine Box I and histidine Box IIIregions of A. thaliana Δ12-desaturase FAD2 (P46313), Caenorhabditiselegans Δ12-desaturase FAT-2 (AAF63745) and Δ15-desaturase FAT-1(L41807), corresponding to LFCVCHDCGH (amino acid residues 88-97) andITNGHVAHH (amino acid residues 291-299) of FAT-2. However, RT-PCRreactions using A. domesticus fat body total RNA with this set ofprimers failed to amplify any specific product despite repeated attemptsand varied conditions, suggesting that the A. domesticus gene(s)encoding Δ12-desaturase might share low homology to the plant ornematode Δ12-desaturase genes.

A different approach was considered to clone the A. domesticusΔ12-desaturase gene. Based on the amino acid sequence homology sharedamong 55 insect fatty acid acyl-CoA Δ9-, Δ10-, Δ11- and Δ14-desaturasesfrom species including Argyrotaenia velutinana (AAF44709), Bombyx mori(BAD18122, AAF80355), Epiphyas postvittana (AAK94070), Helicoverpaassult (AAM28484, AAM28483), H. zea (AAF81787, AAF81788), Muscadomestica (AAN31393), Ostrinia furnacalis (AAL27034, AAL32060,AAL35746), O. nubilalis (AAF44710, AAL35330, AAL35331), Trichoplusia ni(AAB92583, O44390, AAD03775) etc, degenerate primers (AdD12Des-F25′-TTCTTCTTCKCNCAYKTHGGNTGG (SEQ ID NO: 129) andAdD12Des-R25′-TGRTGGTAGTTGTGVHANCCCTC (SEQ ID NO: 130)) were designedthat targeted two conserved regions of these desaturases (FFFS/AHI/VGW(SEQ ID NO: 131) and EGY/W/FHNYHH (SEQ ID NO: 132), corresponding toamino acid residues 145-152 and 263-270 of A. velutinana Δ9-desaturaseAAF44709) in an attempt to clone an insect Δ12-desaturase gene.Surprisingly, RT-PCR from A. domesticus fat body total RNA amplified adivergent 360 bp desaturase-like sequence, along with another desaturasegene fragment identical to part of the known A. domesticus Δ9-desaturase(AdD9Des, AF338466). Rapid amplification of cDNA ends using GeneRacerKit (Invitrogen) and sequences internal to the 360 bp fragment resultedin a 1597 bp cDNA sequence that was designated AdD12Des (SEQ ID NO: 133)coding for a peptide of 357 amino acid, residues (SEQ ID NO: 134).

The cDNA sequences of AdD9Des, AdD12Des and Tribdesat10 were each clonedin yeast expression vector pYES2 (Invitrogen), generating plasmidspXZP277, pXZP282 and pYES2-Trib10 respectively. Plasmids pYES2, pXZP277,pXZP282 and pYES2-Trib10 were transformed into Saccharomyces cerevisiaecell of the strain ole1 which contains a Δ9-desaturase knock-outmutation, (Stukey et al., 1990) in order to test for complementation ofthis mutation and therefore indicate Δ9-desaturase activity. Yeast ole1cells needed to be supplied with an unsaturated fatty acid such asC16:1^(Δ9) to maintain growth, unless a Δ9-desaturase is expressedwithin the cells, which was the basis of the complementation test.Transformants in ole1 cells were selected on drop-out media (SD-Ura)agar plate supplied with 0.5 mM C16:1^(Δ9) and 1% NP-40. Yeast ole1transformants carrying the above mentioned plasmids were first grown inYPD media (1% yeast extract, 2% peptone, 2% dextrose), supplied with 0.5mM C16:1^(Δ9) and 1% NP-40 at 30° C. until OD₆₀₀ around 1.0. The sampleswere then adjusted to an OD₆₀₀ of 1.0, diluted in 1:10 series and 1 μLaliquots of each dilution spotted onto YPG agar plates, which was thesame as YPD media except that it contained 2% galactose instead ofdextrose to induce the gene expression from the pYES2 derived vectors.The growth of cells was observed after 2 days at 30° C. Expression of A.domesticus Δ9-desaturase in ole1 cells clearly complemented the ole1phenotype, while expression of A. domesticus AdD12Des did notcomplement, indicating that it was not functioning as a Δ9-desaturase inyeast cells. The ole1 cells expressing the T. castaneum Δ12-desaturasewere shown to grow on YPG plate only in the presence of added C17:1.These data established that the cloned AdD12Des or Tribdesat10 genes didnot encode another member of a Δ9-desaturase family.

These expression plasmids were also transformed into Saccharomycescerevisiae S288C cells, which is a common laboratory yeast strain. Fattyacid methyl esters (FAME) extraction from yeast cells and analysis wereperformed as described by Zhou, et al. (2006). Expression of AdD12Des inS. cerevisiae produced new diene fatty acids C18:2 and C16:2 from C18:1and C16:1, respectively, as demonstrated by gas chromatography (GC) ofyeast fatty acid methyl esters (FIG. 7A) when compared to cells withpYES2 vector only (FIG. 7B), confirmed by the identical retention timeand mass spectrum as the pure C18:2^(Δ9,Δ12) standard in the firstinstance. The double bond positions of the C16:2 and C18:2 products wereconfirmed by GC-mass spectrometry of the 4,4-dimethyloxazolinederivative (Fay and Richli, 1991) to be at the Δ9 and Δ12 positions bythe gap of 12 atomic mass units between m/z 196 and 208, 236 and 248(FIG. 2C). The production of these dienes demonstrated that AdD12Des andTribdesat10 encoded Δ12-desaturases acting on C16:1^(Δ9) or C18:1^(Δ9).

When expressing in cells of yeast strain ole1, T castaneumΔ12-desaturase produced C18:2 from C18:1 at conversion rate of 30.5%,and C16:2 from C16:1 at the conversion rate of 16.0% (Table 11),indicating a preference of the enzyme for the C18 substrate over the C16substrate. There was a concomitant decrease in the level of substratefatty acid in the cells.

TABLE 11 Fatty acid composition (% total fatty acids) of yeast ole1cells expressing Tribolium castaneum desaturase Tribdesat10 in pYES2when fed C16:1 or C18:1 substrates, compared to an empty pYES2 vectorcontrol. Fatty acid composition (% total fatty acids) 12:0 14:0 15:016:0 16:1 17:0 16:2 17:1 18:0 18:1 18:2 pYES2 + 3.0 2.2 0.7 41.7 0.2 0.60 10.9 16.9 23.8 0 C18:1 Tribdesat10 + 3.3 2.3 1.0 43.2 0.1 0.8 0 8.618.0 15.7 6.9 C18:1 pYES2 + 3.9 2.9 0.7 42.5 20.6 0.5 0 9.1 19.4 0.4 0C16:1 Tribdesat10 + 4.2 3.1 0.8 43.6 14.7 0.6 2.8 7.8 21.9 0.4 0 C16:1

The A. domesticus and T. castaneum Δ12-desaturase protein coding regionswere also cloned into a plant expression vector to generate plasmidpXZP375 and pXZP376 respectively, under control of the seed specificpromoter Fp1 (Stalberg et al., 1993). These expression plasmids wereintroduced into tissues of an A. thaliana fad2/fae1 double mutant (Smithet al., 2003) to produce transformed plants via Agrobacteriumtumefaciens as described by Zhou et al., (2006). GC analysis of seedFAMEs was essentially as described above. Shown in Table 12 as anexample, six transgenic lines transformed with the AdD12Des geneencoding the A. domesticus Δ12-desaturase efficiently produced C18:2,with levels obtained of at least 42% of the total fatty acid in theseedoil, and at least 20% C18:3, both of which were produced in the seedfrom C18:1. Since the A. thaliana fad2/fae1 double mutant still had thewild-type Δ15-desaturase (FAD3), this enzyme was presumed to desaturateC18:2, when available, into C18:3. Therefore, the C18:3 produced inthese transgenic lines was also a product of the conversion of C18:1 toC18:2 bp the A. domesticus Δ12-desaturase, and represented A. domesticusΔ12-desaturase product. This result confirmed the Δ12-desaturaseactivity of the cloned gene, and represented the first demonstration ofa recombinant insect Δ12-desaturase in transformed cells.

TABLE 12 Fatty acid composition (% total fatty acids) of transgenicArabidopsis seeds expressing A. domesticus AdD12Des in fad2/fae1 doublemutant. Sample C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1fad2/fae1* 4.9 0.5 2.09 90.21 0.2 2.06 0.0 0.35 EY1 6.33 0.0 2.46 26.6140.07 24.53 Trace Trace EY6 5.27 2.48 85.93 3.74 2.47  0.11 Trace EY72.9 0.0 44.7 29.1 23.3 0.0 EY13 2.3 0.0 43.1 31.0 23.6 0.0 EY15 3.9 0.025.4 46.2 24.6 0.0 EY20 4.16 0.0 0.05 55.81 19.20 21.11 Trace Trace*fad2/fae1 corresponds to the control (untransformed) plants

Phylogenetic analysis of representative acyl-CoA or acyl-lipiddesaturase protein sequences showed that the amino acid sequences ofAdD12Des and Tribdesat10 clustered with other acyl-CoA desaturases(right hand side of FIG. 9; FIG. 10). The accession numbers and speciesfor insect desaturases used in the analysis were: AdD9, Achetadomesticus AAK25796; AdD12, A. domesticus SEQ ID NO: (SEQ ID NO: 135);BmD10-11, Bombyx mori AAF80355; BmD11, B. mori BAD18122; DmD9,Drosophila melanogaster CAB69054; MdD9, Musca domesticus AAN3:1393;OnD9, Ostrinia nubilalis AAF44710; OnD11, O. nubilalis AAL35331; OnD14,O. nubilalis AAL35330; PoD9, Planotortrix octo AAF73073; PoD10, P. octoAAG54077; TcD9A, Tribolium castaneum XP_(—)969607; TcD9B, T. castaneumXP_(—)966962; TcD12, T. castaneum Tribdesat10 (SEQ ID NO: 28); TnD9,Trichoplusia ni AAB92583; TnD11, T. ni O44390; other animal desaturasesAcD12-15, Acanthamoeba castellanii ABK15557; CeD12, Caenorhabditiselegans AAF63745; RnD9, Rattus norvegicus P07308; bacterial desaturasesN36D9, Nostoc sp. 36 CAF18423ΔNoD9, Nostoc sp. PCC 7120 (Anabaenavariabilis) BAA03434; NoD9, Nostoc sp. PCC 7120 BAA03435; SyD9,Synechocystis PC 6803 BAA03982; SyD12, Synechocystis PC 6803 P20388;fungal desaturases LkD12, Lachancea kluyveri BAD08375; MaD9, Mortierellaalpina BAA75927; Y1D12, Yarrowia lipolytica CAJ81209; ScD9,Saccharomyces cerevisiae P21147; plant desaturases AtD9, Arabidopsisthaliana AAK76592; AtD12FAD2 (ER Δ12-desaturase), Arabidopsis thalianaP46313; AtD12_FAD6 (plastid Δ12-desaturase), A. thaliana P46312; PgD9,Picea glauca AAM12238). D9, D10, D11, D12, and D14 designate Δ9-, Δ10-,Δ11-, Δ12-, and Δ14-desaturases, respectively.

AdD12Des and Tribdesat10 showed only low identities (up to 23%) over thefull-length amino acid sequences to bacterial Δ9-desaturases or plant,fungal and animal Δ12-desaturases which formed another major cluster ofpredominantly acyl-lipid desaturases (left hand side of FIG. 4). The lowlevel of identity of AdD12Des and Tribdesat10 with Δ12-desaturases fromother animals, plants or fungi was particularly unexpected. Instead,AdD12Des formed a discrete cluster with insect Δ9-desaturases includingthose of house cricket and red flour beetle suggesting its possibleevolution from Δ9-desaturase genes. The extent of identity in amino acidsequence of AdD12Des to these were 65.2%, 55.2%, 55.5%, 53.5%, 53.5%,55.3%, 59.4% and 53.6%, respectively, to insect Δ9-desaturases from A.domesticus AF338466, D. melanogaster CAB69054, M. domesticus AAN31393,O. nubilalis AAF44710, P. octo AAF73073, T. castaneum XP_(—)969607, T.castaneum XP_(—)966962 and T. ni AAB9258. In particularly, AdD12Des wasrelatively close in sequence to the Δ9-desaturase protein from the samespecies, sharing about 65% amino acid identity.

As Tribdesat10 appeared on a separate branch of the phylogenic tree tothe other acyl CoA desaturases including Tribolium Δ9-desaturases towhich it shared up to 48% identity, it may have evolved independently ofthe insect Δ9-desaturase genes. As shown by the data in FIG. 3, AdD12Desretained low levels of Δ9-desaturation activity on the medium chainsaturated fatty acids, C14:0 and C15:0, when expressed in yeast. FIG. 3Ashows the partial GC graph of fatty acid profile in yeast cellsexpressing pYES2 vector only, while 3B shows fatty acid profile in yeastcells expressing pXZP282, with extra C14:1 and C15:1 peaks. However, nodetectable Δ9-desaturation activity was seen in yeast expressingTribdesat10. These findings suggest that these two insectΔ12-desaturases independently diverged from an ancient desaturase. Itwas also noteworthy that the other animal Δ12-desaturases fromCaenorhabditis elegans and Acanthamoeba castellanii were more closelyrelated to the plant Δ12-desaturases than to insect Δ12-desaturasesAdD12Des and Tribdesat10 (27.2% and 38.6% amino acid sequence identitiesto Arabidopsis thaliana Δ12-desaturase FAD2, respectively, or 11.6% and11.1% amino acid sequence identities to A. domesticus AdD12Des,respectively).

To test whether the cloned A. domesticus Δ12-desaturase used acyl-CoAC18:1 as substrate rather than acyl-PC C18:1, the Δ12-desaturase genefrom A. domesticus in pXZP282 was expressed in yeast S288C cells. TheArabidopsis thaliana Δ12-desaturase gene coding region encoding. FAD2was also cloned into pYES2, resulted in the construct pXZP279, andexpressed in S288C as a control gene, as an example of a plantΔ12-desaturase that was known to be an acyl-PC type enzyme. Cultures ofthe yeast transformants were incubated in the presence of C¹⁴ labelledC18:1 (5×10⁶ dpm) and cells were harvested at 2, 5, and 30 minutes, and1, 6 and 24 hours later. For optimal results, cells were lysed usingglass beads followed by repeated passes through a tissue grinder. Fattyacids bound to CoA were extracted as described by Domergue et al.(2005), converted to FAMEs and separated by argentation-thin layerchromatography (TLC). Oleic acid added to the medium during inductionbecame immediately available in the acyl-CoA, PC and TAG fractions. Itwas observed that Arabidopsis FAD2 accumulated labelled C18:2 product inthe acyl-CoA fraction from minutes onwards. In contrast, conversion oflabelled C18:1 to C18:2 by the cricket desaturase was observed as earlyas 2 minutes after addition of the substrate, indicating that thisenzyme uses acyl-CoA as substrate. Therefore, AdD12Des, and presumablyTribdesat10, were of the acyl-CoA oleoyl Δ12-desaturase type, ratherthan acyl-PC oleoyl Δ12-desaturases. Genes encoding the former class ofΔ12-desaturase have not previously been identified.

Cloning of the insect acyl-CoA Δ12-desaturases described herein isexpected to stimulate the discovery of other novel insectpolyunsaturated fatty acid desaturase genes and provide greater insightinto insect fatty acid metabolism. The role of this gene in insectdevelopment and physiology can be studied in knockout mutants.Characterization of these Δ12-desaturase genes will also shed more lighton the evolution of insect desaturases via ancestral genes. Finally,this will increase our understanding of sequence-substrate typerelationships among fatty acid desaturases, since membrane-associatedinsect Δ12-desaturases are thought to use acyl-CoA substrates (Cripps,et al. 1990), while membrane-associated plant Δ12-desaturases utilizephosphatidylcholine (PC) as a substrate (Stymne and Appelqvist, 1978).

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1-74. (canceled)
 75. A process for producing oil containing unsaturatedfatty acids comprising i) obtaining a plant or part thereof comprisingan acyl-CoA Δ12 desaturase and an increased level of 16:2^(Δ9,Δ12)and/or 18:2^(Δ9,Δ12) fatty acids relative to a corresponding plant orpart thereof lacking the acyl-CoA Δ12 desaturase, and ii) extracting oilfrom the plant or part thereof.
 76. The process of claim 75, whereinstep ii) comprises producing crude oil by cooking, pressing and/orcrushing the plant or part thereof.
 77. The process of claim 76 whichfurther comprises degumming, refining, bleaching and/or deodorizing thecrude oil.
 78. The process of claim 75 which comprises using a solventto extract the crude oil.
 79. The process of claim 75, wherein the plantor part thereof is a seed.
 80. The process of claim 75, wherein theplant is an oilseed.
 81. The process of claim 75, wherein the plant orpart thereof comprises an increased level of 16:2^(Δ9,Δ12) and/or18:2^(Δ9,Δ12) fatty acids which are esterified to CoA relative to acorresponding plant or part thereof lacking the acyl-CoA Δ12 desaturase.